Food Quality and Safety
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DEPARTMENTS
RESEARCH GROUPS
HIGHLIGHTS
Paper published in Food Microbiology
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR. Alejandro Garrido-Maestu,Sarah Azinheiro, Joana Carvalho, Marta Prado. Volume 73, August 2018, Pages 254–263.
Paper published in Microchemical Journal
Novel approach for accurate minute DNA quantification on microvolumetric solutions
JoanaCarvalho,RenatoNegrinho,SarahAzinheiro, AlejandroGarrido-Maestu,JorgeBarros-Velázquez.MartaPrado.Volume 138,May 2018,Pages 540-549.
Published a Research Article in Front. Sustain. Food Syst.
SarahAzinheiro,JoanaCarvalho,MartaPrado and AlejandroGarrido-Maestu. 21 February 2018.
Paper published in Analytica chimica Acta.
JoanaCarvalho,GemaPuertas,JoãoGaspar,
SarahAzinheiro,LorenaDiéguez,
AlejandroGarrido Maestu, ManuelVázquez,
JorgeBarros-Velázquez,SusanaCardoso,MartaPrado.
Volume 1020, 22 August 2018, Pages 30-40
Scientific Opinion !
The EFSA (European Food Safety Authority) has recently published a scientific opinion on an “Updated quantitative risk assessment (QRA) of the BSE risk posed by processed animal protein (PAP)” & We are happy to mention that Our Food Quality & Safety research Group leader Dr.Marta Prado has participated in this activity Find the links at The Wiley Online Library (WOL) & The EFSA website
Published a Research Article in Food Control
Author: Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado
Food Control 99 , pp. 79-83, 2019.
Book Chapter in collaboration with David Tomás Fornés, Lead Scientist in the Microbial and Molecular Analytics group at Nestlé Research Center (Lausanne, Switzerland). !!!!!! Foodborne Bacterial Pathogens
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens In: Bridier A. (eds) Foodborne Bacterial Pathogens. Methods in Molecular Biology, vol 1918. Humana Press, New York, NY
Reliable Nano & Micro-solutions for Food Safety & Quality Analysis
The main objective of the Food & Quality Research Group is the development of analytical approaches based on the combination of molecular biology (mainly DNA based methodology) and nano and microfabrication technology in order to provide the food industry and control laboratories with reliable analytical tools.
Following this objective, the methodology is based on working on very specific analytical needs and on using a modular approach for each of the steps of the analytical process. This approach, help us to evaluate and to choose the best method in each case, to have a sound integrated final product, and at the same time a wide-range of intermediate products that can be used by themselves to solve specific analytical challenges.
Figure 1 summarizes the overall approach and research lines. Our topics of interest involve the detection of foodborne pathogens, the detection of allergenic ingredients in food products and food authenticity.
RESEARCH LINES
- Sample preparation:
Sample preparation is the series of steps required to transform a sample to a form suitable for analysis, the reliability of the conclusions drawn from food analysis greatly depends upon on this step. We work on: (i) the development of pre-treatment steps in order to overcome some of the limitations associated with food analysis and (ii) on the development of tailored, miniaturized, automatized and faster sample preparation techniques. Microscale solid phase extraction (µSPE) is used for on-chip DNA extraction and purification, being possible to put in contact a higher volume of initial binding material with the solid phase and recover the DNA in a lower volume during the elution phase. This feature allows to concentrate the DNA when minute amounts are present in the sample (e.g. olive oil, wine), for complex matrixes such as processed foodstuff and for environmental samples (e.g. water samples).
- Alternative DNA amplification methods:
Food & Quality Safety Research Group is working on new amplification techniques, in their combination with NPs and on the evaluation of DNA based analytical methods for food analysis. We work on isothermal amplification techniques, such as Loop-Mediated Isothermal Amplification (LAMP), and Recombinase Polymerase Amplification (RPA), specially interesting for miniaturization purposes. Other alternative techniques currently being used include Ligation Chain Reaction (LCR) which allows to distinguish very closely related organisms and high similar DNA sequences.
- Nanoparticle-assisted DNA analysis:
The use of nanomaterials for DNA analysis has the potential of providing increased sensitivity, multiplexing capabilities, and reduced costs. Exploiting the features of nanoparticles (NPs) is considered to be a good alternative to foster the potential of diagnostics and analytical method development. NPs, such as gold NPs (AuNPs) and gold nanorods (AuNRs) are being used for DNA detection taking advantage of their optical properties.
Food Quality and Safety research team (picture from April 2019)
FUNDED PROJECTS
DEVELOPMENT OF A SYSTEM OF EARLY DETECTION OF THE ZEBRA MUSSEL THROUGH ANALYSIS OF ENVIRONMENTAL DNA
Cooperation Agreement with Confederación hidrográfica del Guadalquivir (CHG) Spain.
The objective of this cooperation agreement is the development of a micro Total Analysis Systems (µTAS) and optimized protocol for the detection of zebra mussel, an invasive species affecting numerous river basins worldwide, through environmental DNA (eDNA).
NANOEATERS: Valorization and transfer of NANOtechnologies to EArly adopTERS of the Euroregion Galicia-Norte Portugal-Use Case 2: Olive Oil Characterization
The FQ&S research group participates on the NANOEATERS project, on the Use Case 2: Olive Oil characterization on collaboration with the University of Vigo (Spain).
The main objective of use case 2 is the characterization of Extra virgin olive oil produced in Galicia, and the development of analytical approaches that would allow the differentiation of such olive oil from the one produced elsewhere.
N2020- NBFS-FOODSAFE
Innovative sample preparation and detection methods for foodborne pathogens, allergenic ingredients and food authenticity assessment are being developed. In parallel, new smart packaging and delivery systems are being designed and validated.
PORTGRAPHE
Control of Port and Douro Wines authenticity using graphene DNA sensors project
Time Frame: June 2018 to June 2021
The main objective of the project is the development, test and in-house validation of a miniaturized DNA sensing device for varietal discrimination of grapes, wines, musts, and grape juice in order to ensure the authenticity of wine from Port and Douro DOP. With this objective the participating teams will combine their expertise for the development of a miniaturized analytical device composed of 3 modules namely: a DNA extraction and purification module, an isothermal DNA amplification module, and a DNA Biosensors based on field-effect transistors (FETs) made using single layer graphene (SLG) for varietal discrimination.
NanoBioSensor
Development of nanosensors to evaluate the microbiological quality of fruit-based products
Time Frame: July 2018 to June 2021
A micro-total analysis system will be developed by the participating team to enable faster and better control of selected fruit-based products, reducing the analysis time from 7 days to few hours with greater sensitivity than conventional methods. This device will be designed and manufactured combining the latest developments in molecular biology, microfluidics and electronics.
SF4SF Smart Factoy for Safe Foods
Project funded by CDTI, Ministry of Industry of Spain.
The project aims the integration of emerging technologies for the detection, removal and food hazards management in food processing plants. The role of the group is the development of analytical methods based on DNA detection from gluten producing cereals and Listeria monocytogenes in fish products.
PUBLICATIONS
-
2021
A, Purwidyantri; T, Domingues; J, Borme; R, Guerreiro J; A, Ipatov; M, Abreu C; M, Martins; P, Alpuim; M, Prado
Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors Journal Article
Biosensors, 11 , pp. 24, 2021.
@article{A2021,
title = {Influence of the Electrolyte Salt Concentration on DNA Detection with Graphene Transistors},
author = {Purwidyantri A and Domingues T and Borme J and Guerreiro J R and Ipatov A and Abreu C M and Martins M and Alpuim P and Prado M},
url = {https://doi.org/10.3390/bios11010024},
doi = {10.3390/bios11010024},
year = {2021},
date = {2021-01-17},
journal = {Biosensors},
volume = {11},
pages = {24},
abstract = {Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Liquid-gated Graphene Field-Effect Transistors (GFET) are ultrasensitive bio-detection platforms carrying out the graphene’s exceptional intrinsic functionalities. Buffer and dilution factor are prevalent strategies towards the optimum performance of the GFETs. However, beyond the Debye length (λD), the role of the graphene-electrolytes’ ionic species interactions on the DNA behavior at the nanoscale interface is complicated. We studied the characteristics of the GFETs under different ionic strength, pH, and electrolyte type, e.g., phosphate buffer (PB), and phosphate buffer saline (PBS), in an automatic portable built-in system. The electrostatic gating and charge transfer phenomena were inferred from the field-effect measurements of the Dirac point position in single-layer graphene (SLG) transistors transfer curves. Results denote that λD is not the main factor governing the effective nanoscale screening environment. We observed that the longer λD was not the determining characteristic for sensitivity increment and limit of detection (LoD) as demonstrated by different types and ionic strengths of measuring buffers. In the DNA hybridization study, our findings show the role of the additional salts present in PBS, as compared to PB, in increasing graphene electron mobility, electrostatic shielding, intermolecular forces and DNA adsorption kinetics leading to an improved sensitivity.Jon Ashley Anna Lisa Schaap Johansen, Mohsen Mohammadniaei Maryam Naseri Paolo Marcatili Marta Prado Yi Sun;
Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides Journal Article
Nucleic Acids Research, 2021.
@article{and Annaand Mohsenand MaryamNaseri2021,
title = {Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides},
author = {Jon Ashley, Anna Lisa Schaap Johansen, Mohsen Mohammadniaei, Maryam Naseri, Paolo Marcatili, Marta Prado, Yi Sun;},
url = { https://doi.org/10.1093/nar/gkaa1263},
doi = {10.1093/nar/gkaa1263},
year = {2021},
date = {2021-01-04},
journal = {Nucleic Acids Research},
abstract = {Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets. -
2020
Garrido-Maestu, A; Azinheiro, S; Roumani, F; Carvalho, J; Prado, M
Front. Microbiol, 11 , pp. 1-8, 2020.
@article{Prado2020b,
title = {Application of Short Pre-enrichment, and Double Chemistry Real-Time PCR, Combining Fluorescent Probes and an Intercalating Dye, for Same-Day Detection and Confirmation of Salmonella spp. and Escherichia coli O157 in Ground Beef and Chicken Samples},
author = {A Garrido-Maestu and S Azinheiro and F Roumani and J Carvalho and M Prado},
url = {https://doi.org/10.3389/fmicb.2020.591041},
doi = {10.3389/fmicb.2020.591041},
year = {2020},
date = {2020-10-09},
journal = {Front. Microbiol},
volume = {11},
pages = {1-8},
abstract = {Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Molecular methods, particularly those based on real-time PCR (qPCR), have become a popular approach to detect pathogens in food samples. This technique may take advantage of hydrolysis fluorescent probes for increased specificity. Even though suitable, this approach loses the capacity of performing result confirmation by melt curve analysis. In the current study, we developed an alternative approach, combining fluorescent probes along with an intercalating dye (SYBR Green) in order to simultaneously detect, and confirm the result, of two foodborne pathogens (Salmonella spp. and Escherichia coli O157). This new approach named double chemistry qPCR was combined with a short pre-enrichment in order to obtain a multiplex “same-day” detection method for the selected pathogens. The evaluation of the novel method in spiked food samples (ground beef and chicken breast) obtained values of relative sensitivity, specificity, and accuracy higher than 95%, and Cohen’s kappa of 0.92, with a Limit of Detection95 below 5 cfu/25 g, demonstrating its reliability. In addition to this, the method was challenged by inoculating heat-stressed bacteria as well as dead ones. It was observed that it was also possible to detect stressed bacteria with an initial inoculation level below 10 cfu/25 g. Also, it was noticed that high initial concentration of either pathogen (higher than 104 cfu/25 g) was needed in order to generate false positive results due to the presence of dead bacteria, thus the method presents potential for its application in the specific detection of live microorganisms.Wu, L; Teixeira, A; Garrido-Maestu, A; Muinelo-Romay, L; Lima, L; Santos, LL; Prado, M; Diéguez, L
Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification Journal Article
Biosens. Bioelectron, 165 , 2020.
@article{Wu2020,
title = {Profiling DNA mutation patterns by SERS fingerprinting for supervised cancer classification},
author = {L Wu and A Teixeira and A Garrido-Maestu and L Muinelo-Romay and L Lima and LL Santos and M Prado and L Diéguez},
url = {https://doi.org/10.1016/j.bios.2020.112392},
doi = {10.1016/j.bios.2020.112392},
year = {2020},
date = {2020-10-01},
journal = {Biosens. Bioelectron},
volume = {165},
abstract = {Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Profiling DNA mutation patterns for cancer classification plays an essential role in precision and personalized medicine. Conventional PCR-based mutation assay is limited by the extensive labour on target amplification. We herein create an amplification-free surface enhanced Raman spectroscopy (SERS) biochip which enables direct and simultaneous identification of multiple point mutations in tumor cells. Without pre-amplifying the target sequences, the SERS assay reads out the presence of cellular mutations through the interpretation of Raman fingerprints. The SERS sensor is integrated into a microfluidic chip, achieving one-step multiplex analysis within 40 min. Importantly, by combining SERS spectra encoding technique with supervised learning algorithm, a panel of nucleotide mixtures can be well distinguished according to their mutation profiles. We initially demonstrate an excellent levels of classification in samples from colorectal cancer and melanoma cell lines. For final clinical validation, the system performance is verified by classifying cancer patient samples, which shows an accuracy above 90%. Due to the simplicity and rapidness, the SERS biosensor is expected to become a promising tool for clinical point-of-care diagnosis towards precision medicine.Azinheiro, S; Carvalho, J; Prado, M; Garrido-Maestu, A
Foods, 9 , 2020.
@article{Azinheiro2020,
title = {Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces},
author = {S Azinheiro and J Carvalho and M Prado and A Garrido-Maestu},
url = {https://doi.org/10.3390/foods9091249},
doi = {10.3390/foods9091249},
year = {2020},
date = {2020-09-07},
journal = {Foods},
volume = {9},
abstract = {The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.Azinheiro, S; Carvalho, J; Prado, M
Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula Journal Article
Microorganisms, 8 , 2020.
@article{Azinheiro2020c,
title = {Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula},
author = {S Azinheiro and J Carvalho and M Prado},
url = {https://doi.org/10.3390/microorganisms8091359},
doi = {10.3390/microorganisms8091359},
year = {2020},
date = {2020-09-04},
journal = {Microorganisms},
volume = {8},
abstract = {Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Food poisoning continue to be a threat in the food industry showing a need to improve the detection of the pathogen responsible for the hospitalization cases and death. DNA-based techniques represent a real advantage and allow the detection of several targets at the same time, reducing cost and time of analysis. The development of new methodology using SYBR Green qPCR for the detection of L. monocytogenes, Salmonella spp. and E. coli O157 simultaneously was developed and a non-competitive internal amplification control (NC-IAC) was implemented to detect reaction inhibition. The formulation and supplementation of the enrichment medium was also optimized to allow the growth of all pathogens. The limit of detection (LoD) 95% obtained was <1 CFU/25 g for E. coli O157, and 2 CFU/25 g for Salmonella spp. and L. monocytogenes and regarding the multiplex detection a LoD 95% of 1.7 CFU/25 g was observed. The specificity, relative sensitivity and accuracy of full methodology were 100% and the use of the NC-IAC allowed the reliability of the results without interfering with the sensitivity of the methodology. The described study proved to obtain results comparable to those of probe-based qPCR, and more economically than classical high resolution melting qPCR, being both important aspects for its implementation in the food industry.Oliveira, AJN; de Wild, J; Oliveira, K; Valença, BA; Teixeira, JP; Guerreiro, JRL; Abalde-Cela, S; Lopes, TS; Ribeiro, RM; Cunha, JM V; Curado, MA; Monteiro, M; Violas, A; Silva, AG; Prado, M; Fernandes, PA; Vermang, B; Salomé, PMP
Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells Journal Article
Sol. RRL. , 4 , 2020.
@article{Oliveira2020,
title = {Encapsulation of Nanostructures in a Dielectric Matrix Providing Optical Enhancement in Ultrathin Solar Cells},
author = {AJN Oliveira and J de Wild and K Oliveira and BA Valença and JP Teixeira and JRL Guerreiro and S Abalde-Cela and TS Lopes and RM Ribeiro and JM V Cunha and MA Curado and M Monteiro and A Violas and AG Silva and M Prado and PA Fernandes and B Vermang and PMP Salomé},
url = { https://doi.org/10.1002/solr.202000310},
doi = {10.1002/solr.202000310},
year = {2020},
date = {2020-08-29},
journal = {Sol. RRL. },
volume = {4},
abstract = {The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The incorporation of nanostructures in optoelectronic devices for enhancing their optical performance is widely studied. However, several problems related to the processing complexity and the low performance of the nanostructures have hindered such actions in real‐life devices. Herein, a novel way of introducing gold nanoparticles in a solar cell structure is proposed in which the nanostructures are encapsulated with a dielectric layer, shielding them from high temperatures and harsh growth processing conditions of the remaining device. Through optical simulations, an enhancement of the effective optical path length of approximately four times the nominal thickness of the absorber layer is verified with the new architecture. Furthermore, the proposed concept in a Cu(In,Ga)Se2 solar cell device is demonstrated, where the short‐circuit current density is increased by 17.4%. The novel structure presented in this work is achieved by combining a bottom‐up chemical approach of depositing the nanostructures with a top‐down photolithographic process, which allows for an electrical contact.Azinheiro, S; Kant, K; Shahbazi, MA; Garrido-Maestu, A; Prado, M; L Diéguez,
A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens Journal Article
Food Control, 114 , 2020.
@article{Azinheiro2020b,
title = {A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens},
author = {S Azinheiro and K Kant and MA Shahbazi and A Garrido-Maestu and M Prado and L Diéguez, },
url = {https://doi.org/10.1016/j.foodcont.2020.107242},
doi = {10.1016/j.foodcont.2020.107242},
year = {2020},
date = {2020-08-01},
journal = {Food Control},
volume = {114},
abstract = {Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.A, Garrido-Maestu; S, Azinheiro; J, Carvalho; B, Espiña; M, Prado
Food Sci. Technol., (57), pp. 4143–4151, 2020.
@article{Prado2020,
title = {Evaluation and implementation of commercial antibodies for improved nanoparticle-based immunomagnetic separation and real-time PCR for faster detection of Listeria monocytogenes},
author = {Garrido-Maestu A and Azinheiro S and Carvalho J and Espiña B and Prado M},
url = {https://doi.org/10.1007/s13197-020-04450-1},
doi = {10.1007/s13197-020-04450-1},
year = {2020},
date = {2020-04-24},
journal = {Food Sci. Technol.},
number = {57},
pages = {4143–4151},
abstract = {L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}L. monocytogenes continues to be a major health issue in Europe, as well as worldwide. Faster methods, not only for detection, but also for sample preparation are of great interest particularly for this slow-growing pathogen. Immunomagnetic separation has been previously reported to be an effective way to concentrate bacteria, and remove inhibitors. In the present study, different commercial antibodies were evaluated to select the most appropriate one, in order to develop a highly specific method. Additionally, magnetic nanoparticles, instead of microparticles, were selected due to their reported advantages (higher surface-volume ration and faster kinetics). Finally, the separation protocol, with a calculated capture efficiency of 95%, was combined with real-time PCR for highly sensitive detection of the concentrated bacteria. The optimized IMS-qPCR allowed to reduce hands-on time in the sample treatment, without affecting the overall performance of the method as a very low limit of detection was still obtained (9.7 CFU/ 25 g) with values for sensitivity, specificity, accuracy, positive and negative predictive values of 100%, resulting in a kappa index of concordance of 1.00. These results were obtained in spiked food samples of different types (chicken, fish, milk, hard and fresh cheese), further demonstrating the applicability of the optimized methodology presented.Teixeira, A; Paris, JL; Roumani, F; Diéguez, L; Prado, M; Espiña, B; Abalde-Cela, S; Garrido-Maestu, A; Rodriguez-Lorenzo, L
Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach Journal Article
Materials (Basel), 13 , 2020.
@article{Teixeira2020,
title = {Multifuntional gold nanoparticles for the SERS detection of pathogens combined with a LAMP-in-microdroplets approach},
author = {A Teixeira and JL Paris and F Roumani and L Diéguez and M Prado and B Espiña and S Abalde-Cela and A Garrido-Maestu and L Rodriguez-Lorenzo},
url = {https://doi.org/10.3390/ma13081934},
doi = {10.3390/ma13081934},
year = {2020},
date = {2020-04-20},
journal = {Materials (Basel)},
volume = {13},
abstract = {We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}We developed a droplet-based optofluidic system for the detection of foodborne pathogens. Specifically, the loop-mediated isothermal amplification (LAMP) technique was combined with surface-enhanced Raman scattering (SERS), which offers an excellent method for DNA ultradetection. However, the direct SERS detection of DNA compromises the simplicity of data interpretation due to the variability of its SERS fingerprints. Therefore, we designed an indirect SERS detection method using multifunctional gold nanoparticles (AuNPs) based on the formation of pyrophosphate generated during the DNA amplification by LAMP. Towards this goal, we prepared multifunctional AuNPs involving three components with key roles: (1) thiolated poly(ethylene glycol) as stabilizing agent, (2) 1-naphthalenethiol as Raman reporter, and (3) glutathione as a bioinspired chelating agent of magnesium (II) ions. Thus, the variation in the SERS signal of 1-naphthalenethiol was controlled by the aggregation of AuNPs triggered by the complexation of pyrophosphate and glutathione with free magnesium ions. Using this strategy, we detected Listeria monocytogenes, not only in buffer, but also in a food matrix (i.e., ultra-high temperaturemilk) enabled by the massive production of hotspots as a result of the self-assemblies that enhanced the SERS signal. This allowed the development of a microdroplet-LAMP-SERS platform with isothermal amplification and real-time identification capabilities.Garrido-Maestu, A; Azinheiro, S; Carvalho, J; Fuciños, P; Prado, M
Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens Journal Article
Food Control, 108 , 2020.
@article{Garrido-Maestu2020,
title = {Optimized sample treatment, combined with real-time PCR, for same-day detection of E. coli O157 in ground beef and leafy greens},
author = {A Garrido-Maestu and S Azinheiro and J Carvalho and P Fuciños and M Prado},
url = {https://doi.org/10.1016/j.foodcont.2019.106790},
doi = {10.1016/j.foodcont.2019.106790},
year = {2020},
date = {2020-02-01},
journal = {Food Control},
volume = {108},
abstract = {E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in “next-day detection” methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real “same-day detection” method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in “almost complete concordance” with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods. -
2019
Rodriguez-Lorenzo, L; Garrido-Maestu, A; Bhunia, AK; Espiña, B; Prado, M; Diéguez, L; Abalde-Cela, S
Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics Journal Article
ACS Appl. Nano Mater., 2 (10), pp. 6081–6086, 2019.
@article{Abalde-Cela2019,
title = {Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics},
author = {L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela},
url = {https://doi.org/10.1021/acsanm.9b01223},
doi = {10.1021/acsanm.9b01223},
year = {2019},
date = {2019-09-20},
journal = {ACS Appl. Nano Mater.},
volume = {2},
number = {10},
pages = {6081–6086},
abstract = {Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.Alejandro Garrido-Maestu Sarah Azinheiro, Carla Carvalho Joana Carvalho Marta Prado
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019b,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Carla Carvalho, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.Alejandro Garrido-Maestu PabloFuciños, SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
Lei Wu Alejandro Garrido-Maestu, Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Lei Wu , Alejandro Garrido-Maestu , Joana R. L. Guerreiro , Sandra Carvalho , Sara Abalde-Cela , Marta Prado, Lorena Diéguez},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care. -
2018
Alejandro Garrido-Maestu David Tomás Fornés, Marta Prado Rodríguez
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018b,
title = { The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Alejandro Garrido-Maestu, David Tomás Fornés, Marta Prado Rodríguez},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.JoanaCarvalho RenatoNegrinho, SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018b,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {JoanaCarvalho,RenatoNegrinho,SarahAzinheiro,AlejandroGarrido-Maestu,JorgeBarros-Velázquez,MartaPrado},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.JoanaCarvalho GemaPuertas, JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoanaCarvalho,GemaPuertas,JoãoGaspar,SarahAzinheiro,LorenaDiéguez,AlejandroGarrido-Maestu,ManuelVázquez,JorgeBarros-Velázquez,SusanaCardoso,MartaPradoa},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.Alejandro Garrido-Maestu, Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017b,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Alejandro Garrido-Maestu, , Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Pablo Fuciños Marta Prado
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017b,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Joana Carvalho, Pablo Fuciños, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Marta Prado
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = { Alejandro Garrido-Maestu,Sarah Azinheiro, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Sarah Azinheiro Joana Carvalho, Marta Prado ; Garrido-Maestu., Alejandro
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = { Sarah Azinheiro, Joana Carvalho, Marta Prado and Alejandro Garrido-Maestu.},
editor = {Joshua B. Gurtler, Agricultural Research Service (USDA), United States },
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005 },
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods },
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsJoana Carvalho Renato Negrinho, Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Joana Carvalho, Renato Negrinho, Sarah Azinheiro, Alejandro Garrido-Maestu, Jorge Barros-Velázquez, Marta Prado},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
-
2017
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017b,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Joana Carvalho, Sara Abalde-Cela, Enrique Carbó-Argibay, Lorena Dieguez, Marek Piotrowski, Yury V. Kolen'ko, Marta Prado},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Garrido-Maestu A.; Fuciños, Azinheiro Carvalho Prado P ; S ; J ; M
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Garrido-Maestu, A.; Fuciños, P.; Azinheiro, S.; Carvalho, J.; Prado, M.},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Vial S.; Berrahal, Prado Wenger Y ; M ; J
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors , 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Vial, S.; Berrahal, Y.; Prado, M.; Wenger, J.},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors },
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
-
2016
Prado M.; Espiña, Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K
Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends Book Chapter
Antimicrobial Food Packaging, pp. pp.183-202, Jorge Barros-Velazquez, 1st Edition, 2016, ISBN: 978-0-12-800723-5.
@inbook{Prado2016b,
title = {Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends},
author = {Prado, M.; Espiña, B.; Fernandez-Arguelles, M. T.; Diéguez, L.; Fuciños, P.; Vial, S.; Oliveira, J. M.; Reis, R. L.; Boehme, K.},
url = {https://www.sciencedirect.com/science/book/9780128007235},
isbn = {978-0-12-800723-5},
year = {2016},
date = {2016-12-01},
booktitle = {Antimicrobial Food Packaging},
journal = {Academic Press, London},
pages = {pp.183-202},
publisher = {Jorge Barros-Velazquez},
edition = {1st Edition},
abstract = {Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.
},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.Prado M.; Ortea, Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition , 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Prado, M.; Ortea, I.; Vial, S.; Rivas, J.; Calo-Mata, P.; Barros-Velázquez, J.},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition },
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
GROUP LEADER
THE TEAM
Alejandro Garrido
Staff Researcher
Andrey Ipatov
Research Fellow
Foteini Roumani
Research Fellow
Agnes Purwidyantri
Research Fellow
Sarah Azinheiro
PhD Student
Shambhavi Yadav
Scientific Associate
FORMER GROUP MEMBERS
Carlos Carpena
visitor from Ocupharm diagnostics S.L.
Joana Carvalho
Laboratory Assistant
Joana Guerreiro
Research Fellow
Jon Ashley
Research Fellow
Karola Böhme
Research Fellow
Monisha Elumalai
Research Fellow
Master student ( February – September 2017)
University de Santiago de Compostela
Visitor (June 2017)
Sofia Granja Martins
Summer Student (July – September 2017)
María Leyva (Scientific Visitor)
Anna Toldrà Filella
Visitor from IRTA
Cristina Pastrana
visitor from Ocupharm diagnostics S.L.
NEWS & EVENTS
New funded projects!
-
PORT GRAPHE – Control of Port and Douro Wines authenticity using graphene DNA sensors project(Project Time Frame: June 2018 to June 2021)
-
Nano BioSensor – Development of nanosensors to evaluate the microbiological quality of fruit-based products(Project Time Frame: July 2018 to June 2021)
Reliable Nano & Micro-solutions for Food Safety & Quality Analysis
The main objective of the Food & Quality Research Group is the development of analytical approaches based on the combination of molecular biology (mainly DNA based methodology) and nano and microfabrication technology in order to provide the food industry and control laboratories with reliable analytical tools.
Following this objective, the methodology is based on working on very specific analytical needs and on using a modular approach for each of the steps of the analytical process. This approach, help us to evaluate and to choose the best method in each case, to have a sound integrated final product, and at the same time a wide-range of intermediate products that can be used by themselves to solve specific analytical challenges.
Figure 1 summarizes the overall approach and research lines. Our topics of interest involve the detection of foodborne pathogens, the detection of allergenic ingredients in food products and food authenticity.
RESEARCH LINES
- Sample preparation:
Sample preparation is the series of steps required to transform a sample to a form suitable for analysis, the reliability of the conclusions drawn from food analysis greatly depends upon on this step. We work on: (i) the development of pre-treatment steps in order to overcome some of the limitations associated with food analysis and (ii) on the development of tailored, miniaturized, automatized and faster sample preparation techniques. Microscale solid phase extraction (µSPE) is used for on-chip DNA extraction and purification, being possible to put in contact a higher volume of initial binding material with the solid phase and recover the DNA in a lower volume during the elution phase. This feature allows to concentrate the DNA when minute amounts are present in the sample (e.g. olive oil, wine), for complex matrixes such as processed foodstuff and for environmental samples (e.g. water samples).
- Alternative DNA amplification methods:
Food & Quality Safety Research Group is working on new amplification techniques, in their combination with NPs and on the evaluation of DNA based analytical methods for food analysis. We work on isothermal amplification techniques, such as Loop-Mediated Isothermal Amplification (LAMP), and Recombinase Polymerase Amplification (RPA), specially interesting for miniaturization purposes. Other alternative techniques currently being used include Ligation Chain Reaction (LCR) which allows to distinguish very closely related organisms and high similar DNA sequences.
- Nanoparticle-assisted DNA analysis:
The use of nanomaterials for DNA analysis has the potential of providing increased sensitivity, multiplexing capabilities, and reduced costs. Exploiting the features of nanoparticles (NPs) is considered to be a good alternative to foster the potential of diagnostics and analytical method development. NPs, such as gold NPs (AuNPs) and gold nanorods (AuNRs) are being used for DNA detection taking advantage of their optical properties.
DEVELOPMENT OF A SYSTEM OF EARLY DETECTION OF THE ZEBRA MUSSEL THROUGH ANALYSIS OF ENVIRONMENTAL DNA
Cooperation Agreement with Confederación hidrográfica del Guadalquivir (CHG) Spain.
The objective of this cooperation agreement is the development of a micro Total Analysis Systems (µTAS) and optimized protocol for the detection of zebra mussel, an invasive species affecting numerous river basins worldwide, through environmental DNA (eDNA).
NANOEATERS: Valorization and transfer of NANOtechnologies to EArly adopTERS of the Euroregion Galicia-Norte Portugal-Use Case 2: Olive Oil Characterization
The FQ&S research group participates on the NANOEATERS project, on the Use Case 2: Olive Oil characterization on collaboration with the University of Vigo (Spain).
The main objective of use case 2 is the characterization of Extra virgin olive oil produced in Galicia, and the development of analytical approaches that would allow the differentiation of such olive oil from the one produced elsewhere.
N2020- NBFS-FOODSAFE
Innovative sample preparation and detection methods for foodborne pathogens, allergenic ingredients and food authenticity assessment are being developed. In parallel, new smart packaging and delivery systems are being designed and validated.
PORTGRAPHE
Control of Port and Douro Wines authenticity using graphene DNA sensors project
Time Frame: June 2018 to June 2021
The main objective of the project is the development, test and in-house validation of a miniaturized DNA sensing device for varietal discrimination of grapes, wines, musts, and grape juice in order to ensure the authenticity of wine from Port and Douro DOP. With this objective the participating teams will combine their expertise for the development of a miniaturized analytical device composed of 3 modules namely: a DNA extraction and purification module, an isothermal DNA amplification module, and a DNA Biosensors based on field-effect transistors (FETs) made using single layer graphene (SLG) for varietal discrimination.
NanoBioSensor
Development of nanosensors to evaluate the microbiological quality of fruit-based products
Time Frame: July 2018 to June 2021
A micro-total analysis system will be developed by the participating team to enable faster and better control of selected fruit-based products, reducing the analysis time from 7 days to few hours with greater sensitivity than conventional methods. This device will be designed and manufactured combining the latest developments in molecular biology, microfluidics and electronics.
SF4SF Smart Factoy for Safe Foods
Project funded by CDTI, Ministry of Industry of Spain.
The project aims the integration of emerging technologies for the detection, removal and food hazards management in food processing plants. The role of the group is the development of analytical methods based on DNA detection from gluten producing cereals and Listeria monocytogenes in fish products.
PUBLICATIONS
-
2019
Rodriguez-Lorenzo, L; Garrido-Maestu, A; Bhunia, AK; Espiña, B; Prado, M; Diéguez, L; Abalde-Cela, S
Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics Journal Article
ACS Appl. Nano Mater., 2 (10), pp. 6081–6086, 2019.
@article{Abalde-Cela2019,
title = {Gold Nanostars for the Detection of Foodborne Pathogens via Surface-Enhanced Raman Scattering Combined with Microfluidics},
author = {L Rodriguez-Lorenzo and A Garrido-Maestu and AK Bhunia and B Espiña and M Prado and L Diéguez and S Abalde-Cela},
url = {https://doi.org/10.1021/acsanm.9b01223},
doi = {10.1021/acsanm.9b01223},
year = {2019},
date = {2019-09-20},
journal = {ACS Appl. Nano Mater.},
volume = {2},
number = {10},
pages = {6081–6086},
abstract = {Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Herein, we demonstrated the potential of surface-enhanced Raman scattering (SERS) spectroscopy combined with microfluidics for the detection and discrimination of foodborne pathogens. SERS-tagged gold nanostars were functionalized with a monoclonal antibody specific for Listeria monocytogenes. In the presence of L. monocytogenes, a SERS signal corresponding to the SERS tag paired to the antibody was detected in real time and in continuous flow, enabling the discrimination of L. monocytogenes and Listeria innocua in just 100 s. To the best of our knowledge, this is the first time that SERS tags have been used for the in-flow detection of living organisms.Alejandro Garrido-Maestu Sarah Azinheiro, Carla Carvalho Joana Carvalho Marta Prado
Food Science, 84 (7), pp. 1881-1887, 2019.
@article{Garrido-Maestu2019b,
title = {Combination of Immunomagnetic separation and real-time Recombinase Polymerase Amplification (IMS-qRPA) for specific detection of Listeria monocytogenes in smoked salmon samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Carla Carvalho, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1111/1750-3841.14662},
doi = {10.1111/1750-3841.14662},
year = {2019},
date = {2019-07-02},
journal = {Food Science},
volume = {84},
number = {7},
pages = {1881-1887},
abstract = {Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Nowadays, Listeria monocytogenes continues to be a major health issue. Therefore, improvements in the speed and reliability of its detection are still needed. In the present study, the combination of real‐time Recombinase Polymerase Amplification (qRPA) with immunomagnetic separation (IMS) is described. The proposed methodology was tested against a real‐time PCR method, and was successfully applied to 50 smoked salmon samples spiked at levels ranging from 2 to 9.3 × 102 cfu/25 g. L. monocytogenes was detected after a 24 hr pre‐enrichment, which represents a great improvement over other previously published RPA methods. Additionally, the evaluation of the method reported a Limit of dDetection 50 (LoD50) of 6.3 cfu/25 g, along with relative sensitivity, specificity and accuracy values higher than 90%. Finally, the index of kappa concordance was calculated to be 0.93 which is interpreted as “almost complete concordance” between the reference and alternative method. Overall, the described methodology proved to be faster, specific, and as sensitive as other methods based on RPA or real‐time PCR.Alejandro Garrido-Maestu PabloFuciños, SarahAzinheiro CarlaCarvalho JoanaCarvalho MartaPrado
Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples Journal Article
Food Control, 99 , pp. 79-83, 2019.
@article{Garrido-Maestu2019,
title = {Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples},
author = {Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado},
url = {https://doi.org/10.1016/j.foodcont.2018.12.038},
doi = {10.1016/j.foodcont.2018.12.038},
year = {2019},
date = {2019-05-01},
journal = {Food Control},
volume = {99},
pages = {79-83},
abstract = {Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Serovar Enteritidis represents 45.7% of all Salmonella reported human cases identified in Europe. Additionally, “minced meat and meat preparations from poultry” have a high level of non-compliance, regarding Salmonella regulation.In the current study, a novel method based on the amplification of the Salmonella bacteriophage vB_SenS_PVP-SE2, coupled with real-time PCR (qPCR), was developed and evaluated, for the rapid detection of viable Salmonella Enteritidis in chicken samples. The results obtained indicated that the qPCR method could detect down to 0.22 fg/μL of pure virus DNA and a concentration of viral particles of 103 pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8 cfu/25 g could be detected within 10 h, including the time for DNA extraction and qPCR analysis. Additionally, the evaluation of the performance parameters: relative sensitivity, specificity, accuracy, positive and negative predictive values, and index kappa of concordance, obtained values higher than 92%, and the acceptability limit values were within the limits. All these results demonstrate that the proposed methodology is a powerful tool for the rapid detection of viable Salmonella Enteritidis.
Lei Wu Alejandro Garrido-Maestu, Joana Guerreiro Sandra Carvalho Sara Abalde-Cela Marta Prado Lorena Diéguez R L
Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity Journal Article
Nanoscale, 11 (16), pp. 7781-7789, 2019.
@article{Wu2019,
title = {Amplification-free SERS analysis of DNA mutation in cancer cells with single-base sensitivity},
author = {Lei Wu , Alejandro Garrido-Maestu , Joana R. L. Guerreiro , Sandra Carvalho , Sara Abalde-Cela , Marta Prado, Lorena Diéguez},
url = {https://doi.org/10.1039/C9NR00501C},
doi = {10.1039/C9NR00501C},
year = {2019},
date = {2019-04-01},
journal = {Nanoscale},
volume = {11},
number = {16},
pages = {7781-7789},
abstract = {Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Accurate and sensitive identification of DNA mutations in tumor cells is critical to the diagnosis, prognosis and personalized therapy of cancer. Conventional polymerase chain reaction (PCR)-based methods are limited by the complicated amplification process. Herein, an amplification-free surface enhanced Raman spectroscopy (SERS) approach which directly detects point mutations in cancer cells has been proposed. A highly sensitive and uniform SERS substrate was fabricated using gold@silver core–shell nanorods, achieving an enhancement factor of 1.85 × 106. By combining the SERS-active nanosubstrate with molecular beacon probes, the limit of detection reached as low as 50 fM. To enable parallel analysis and automated operation, the SERS sensor was integrated into a microfluidic chip. This novel chip-based assay was able to differentiate between mutated and wild-type KRAS genes among a variety of other nucleic acids from cancer cells in 40 min. Owing to the simple operation and fast analysis, the SERS-based DNA assay chip could potentially provide insights into clinical cancer theranostics in an easy and inexpensive manner at the point of care. -
2018
Alejandro Garrido-Maestu David Tomás Fornés, Marta Prado Rodríguez
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens Book Chapter
Methods in Molecular Biology, 1918 , pp. 35-45, Humana Press, New York, NY, 2018.
@inbook{Garrido-Maestu2018b,
title = { The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens},
author = {Alejandro Garrido-Maestu, David Tomás Fornés, Marta Prado Rodríguez},
url = {https://doi.org/10.1007/978-1-4939-9000-9_3},
doi = {10.1007/978-1-4939-9000-9_3},
year = {2018},
date = {2018-12-23},
booktitle = {Methods in Molecular Biology},
volume = {1918},
pages = {35-45},
publisher = {Humana Press, New York, NY},
abstract = {Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Foodborne pathogens continue to be a major health issue worldwide. Culture-dependent methodologies are still considered the gold-standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor-intensive. The implementation of real-time PCR has allowed to overcome these limitations and even reduce costs associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.JoanaCarvalho RenatoNegrinho, SarahAzinheiro AlejandroGarrido-Maestu JorgeBarros-Velázquez MartaPrado
Elsevier, 21 (21), pp. 424-431, 2018.
@article{JoanaCarvalho2018b,
title = {Data on minute DNA quantification on microvolumetric solutions: comparison of mathematical models and effect of some compounds on the DNA quantification accuracy},
author = {JoanaCarvalho,RenatoNegrinho,SarahAzinheiro,AlejandroGarrido-Maestu,JorgeBarros-Velázquez,MartaPrado},
url = {https://www.sciencedirect.com/science/article/pii/S2352340918311764},
doi = {https://doi.org/10.1016/j.dib.2018.09.098},
year = {2018},
date = {2018-10-02},
journal = {Elsevier},
volume = {21},
number = {21},
pages = {424-431},
abstract = {This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}This article contains data related to the research article entitled “Novel approach for accurate minute DNA quantification on microvolumetric solutions” (Carvalho et al., 2018). The combination of PicoGreen® with a microvolume fluorospectrometer is a popular DNA quantification method due to its high sensitivity and minimal consumption of sample, being commonly used to evaluate the performance of microfluidic devices designed for DNA purification. In this study, the authors present data related with the effect of DNA fragmentation level. The present data article includes the data used on the precision evaluation, in terms of repeatability, of the mathematical models developed to obtain the standards curve for salmon sperm DNA (low molecular weight). In addition, results related with the effect of some compounds on the DNA quantification accuracy using λDNA are presented.JoanaCarvalho GemaPuertas, JoãoGaspar SarahAzinheiro LorenaDiéguez AlejandroGarrido-Maestu ManuelVázquez JorgeBarros-Velázquez SusanaCardoso MartaPradoa
Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device Journal Article
Analytica Chimica Acta, 1020 , pp. 30-40, 2018.
@article{JoanaCarvalho2018,
title = {Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device},
author = {JoanaCarvalho,GemaPuertas,JoãoGaspar,SarahAzinheiro,LorenaDiéguez,AlejandroGarrido-Maestu,ManuelVázquez,JorgeBarros-Velázquez,SusanaCardoso,MartaPradoa},
url = {https://doi.org/10.1016/j.aca.2018.02.079},
doi = {https://doi.org/10.1016/j.aca.2018.02.079},
year = {2018},
date = {2018-08-22},
journal = {Analytica Chimica Acta},
volume = {1020},
pages = {30-40},
abstract = {Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.Alejandro Garrido-Maestu, Sarah Azinheiro Pablo Fuciños Joana Carvalho Marta Prado
Food Chemistry, Volume 246 , pp. Pages 156–163, 2018.
@article{Garrido-Maestu2017b,
title = {Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR)},
author = {Alejandro Garrido-Maestu, , Sarah Azinheiro, Pablo Fuciños, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.foodchem.2017.11.005},
year = {2018},
date = {2018-04-25},
journal = {Food Chemistry},
volume = {Volume 246},
pages = {Pages 156–163},
abstract = {The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR.Keywords: Real-time Loop-mediated isothermal AMPlification (qLAMP); Real-time PCR (qPCR); α2-gliadin; Gluten; Gluten-free food
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Pablo Fuciños Marta Prado
Food Control, Volume 86 , pp. Pages 27–34, 2018.
@article{Garrido-Maestu2017b,
title = {Development and evaluation of loop-mediated isothermal amplification, and Recombinase Polymerase Amplification methodologies, for the detection of Listeria monocytogenes in ready-to-eat food samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Joana Carvalho, Pablo Fuciños, Marta Prado},
url = {https://doi.org/10.1016/j.foodcont.2017.11.006},
year = {2018},
date = {2018-04-01},
journal = {Food Control},
volume = {Volume 86},
pages = {Pages 27–34},
abstract = {Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeriosis continues to be a major health issue. This is demonstrated by the fact that, even though efforts have been made, its incidence does not decrease. Furthermore, in Europe, over 2014 a 30% increase was reported respect to 2013. In the present study two isothermal DNA amplification methods, one based on Loop-mediated isothermal AMPlification (qLAMP), and the other on Recombinase Polymerase Amplification (RPA), were developed and extensively evaluated. Both techniques demonstrated their reliability to detect Listeria monocytogenes in different types of foods. The method included a two-step enrichment, which additionally reduces the chances of detecting dead bacteria. Over the evaluation with pure bacterial DNA, the qLAMP and RPA methods resulted 10 to 100 times less sensitive than qPCR (with two different detection chemistries), but when tested in real food samples the results showed very good concordance with those obtained by qPCR and by selective agar plating (index kappa of concordance between 0.90 and 0.95). Additionally, a very low limit of detection (below 10 CFU/25 g) was obtained. Thus the optimal performance of both isothermal techniques, and their adequacy for their implementation in the food industry, was demonstrated.Keywords: qLAMP; RPA; qPCR; hly; Listeria monocytogenes
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Marta Prado
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR Journal Article
Food Microbiology, 73 , pp. 254-263, 2018.
@article{Garrido-Maestu2018,
title = {Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR},
author = { Alejandro Garrido-Maestu,Sarah Azinheiro, Joana Carvalho, Marta Prado},
url = {https://doi.org/10.1016/j.fm.2018.02.004},
year = {2018},
date = {2018-02-07},
journal = {Food Microbiology},
volume = {73},
pages = {254-263},
abstract = {Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Listeria monocytogenes continues to be one of the most important foodborne pathogens worldwide, either due to its incidence and/or to its high mortality rate. In the present study, a filtration-based protocol was applied for the screening of viable bacteria. Additionally, a complete method (enrichment, filtration, DNA extraction and real-time PCR detection) was evaluated in order to determine the capacity of this protocol to detect viable L. monocytogenes in food samples. A new multiplex qPCR detection system was designed, including an internal amplification control, both targets were detected with hydrolysis probes. It was demonstrated that the method could reliably detect this pathogen, reaching a limit of detection of 9.5 cfu/25 g. The evaluation of the relative sensitivity, specificity, accuracy, positive and negative predictive values, as well as the index kappa of concordance obtained values higher than 90.0% after 24 h sample enrichment. Furthermore, it was demonstrated that with a secondary enrichment step, the limit of the detection could be further decreased to 4.6 cfu/25 g without significantly affecting the performance parameters. The present study demonstrates the reliability of the proposed methodology for the detection of viable L. monocytogenes, and the possibility of its direct implementation for routine analyses in the food industry.Sarah Azinheiro Joana Carvalho, Marta Prado ; Garrido-Maestu., Alejandro
Frontiers in sustainable food systems, 2018.
@article{Azinheiro2018,
title = {Evaluation of different genetic targets for Salmonella enterica serovar Enteriditis and Typhimurium, using Loop-mediated isothermal AMPlification for detection in food samples},
author = { Sarah Azinheiro, Joana Carvalho, Marta Prado and Alejandro Garrido-Maestu.},
editor = {Joshua B. Gurtler, Agricultural Research Service (USDA), United States },
url = {https://www.frontiersin.org/articles/10.3389/fsufs.2018.00005/abstract},
doi = {doi: 10.3389/fsufs.2018.00005 },
year = {2018},
date = {2018-02-05},
journal = {Frontiers in sustainable food systems},
abstract = {Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methods },
keywords = {},
pubstate = {published},
tppubtype = {article}
}Salmonella Enteritidis and Salmonella Typhimurium continue to be the most frequently identified serovars among confirmed cases of salmonellosis. In the current study different genetic targets (safA, sdf I, STM4497 and typh) were compared, attending to their specificity and sensitivity in pure cultures and in spiked samples, in order to determine their capacity to accurately identify them by loop-mediated isothermal amplification (LAMP). For the genes selected to detect Enteritidis, both performed equally well regarding their specificity, but safA proved more sensitive than Sdf I; minor differences were observed among these genes when analyzing spiked food samples. Regarding the targets for Typhimurium, STM4497 and typh, the former demonstrated to be more specific and sensitive, both when analyzing pure cultures as well as spiked samples. These results highlight the importance of an adequate evaluation of the genetic targets selected, before their implementation for routine analyses.
Keywords: Salmonella enteritidis, SAFA, Sdf I, Salmonella typhimurium, STM4497, typh, LAMP, Characterization methodsJoana Carvalho Renato Negrinho, Sarah Azinheiro Alejandro Garrido-Maestu Jorge Barros-Velázquez Marta Prado
Novel approach for accurate minute DNA quantification on microvolumetric solutions Journal Article
Microchemical Journal, 2018.
@article{Carvalho2018,
title = {Novel approach for accurate minute DNA quantification on microvolumetric solutions},
author = {Joana Carvalho, Renato Negrinho, Sarah Azinheiro, Alejandro Garrido-Maestu, Jorge Barros-Velázquez, Marta Prado},
url = {https://doi.org/10.1016/j.microc.2018.02.001},
year = {2018},
date = {2018-02-03},
journal = {Microchemical Journal},
abstract = {The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE},
keywords = {},
pubstate = {published},
tppubtype = {article}
}The optimization and evaluation of the performance of microfluidic devices for DNA purification requires the use of a reliable DNA quantification method. The samples collected from these devices usually have small volumes and, in the case of food, forensic and environmental applications, these samples are also complex, frequently containing highly fragmented DNA and minute concentrations. Therefore, combining a PicoGreen® assay with a microvolume fluorospectrometer is a popular technique for DNA quantification, providing highly sensitive quantification with minimal consumption of sample. However, this method has limitations, such as being affected by the degree of fragmentation of DNA and by the presence of some compounds commonly used in DNA extraction and purification protocols. In this work, these limitations and their influence on the accuracy of the quantification method were evaluated. Low molecular weight salmon sperm DNA was selected, being less purified and more fragmented than the λDNA standard most frequently used. It was shown that the standard curves generated with λDNA were not suitable for the quantification of fragmented DNA, such as DNA from highly processed samples and/or samples that have been exposed to harsh environments. In addition, a mathematical model was developed to find a better adjustment for the standard curve, for the salmon sperm DNA samples. This approach can be used as a tool to overcome important limitations found in this quantification method, allowing to include more data in the standard curve or test different mathematical models to better fit the standards data.Keywords
DNA quantification; PicoGreen; μTAS; DNA analysis; DNA purification; μSPE
-
2017
Alejandro Garrido-Maestu Sarah Azinheiro, Joana Carvalho Sara Abalde-Cela Enrique Carbó-Argibay Lorena Dieguez Marek Piotrowski Yury Kolen'ko Marta Prado V
Frontiers in Microbiology In Press, 2017.
@article{Garrido-Maestu2017b,
title = {Combination of microfluidic loop-mediated isothermal amplification with gold nanoparticles for rapid detection of Salmonella spp. in food samples},
author = {Alejandro Garrido-Maestu, Sarah Azinheiro, Joana Carvalho, Sara Abalde-Cela, Enrique Carbó-Argibay, Lorena Dieguez, Marek Piotrowski, Yury V. Kolen'ko, Marta Prado},
url = {https://www.frontiersin.org/articles/10.3389/fmicb.2017.02159/abstract},
doi = {doi: 10.3389/fmicb.2017.02159},
year = {2017},
date = {2017-10-20},
journal = {Frontiers in Microbiology In Press},
abstract = {Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Foodborne diseases are an important cause of morbidity and mortality. According to the World Health Organization, there are 31 main global hazards, which caused in 2010 600 million foodborne illnesses and 420000 deaths. Among them, Salmonella spp. is one of the most important human pathogens, accounting for more than 90000 cases in Europe and even more in the United States per year. In the current study we report the development, and thorough evaluation in food samples, of a microfluidic system combining loop-mediated isothermal amplification with gold nanoparticles (AuNPs). This system is intended for low-cost, in-situ, detection of different pathogens, as the proposed methodology can be extrapolated to different microorganisms. A very low limit of detection (10 cfu/ 25 g) was obtained. Furthermore, the evaluation of spiked food samples (chicken, turkey, egg products), completely matched the expected results, as denoted by the index kappa of concordance (value of 1.00). The results obtained for the relative sensitivity, specificity and accuracy were of 100 % as well as the positive and negative predictive values.Garrido-Maestu A.; Fuciños, Azinheiro Carvalho Prado P ; S ; J ; M
Food Control, 80 , pp. Pages 297-306, 2017.
@article{Garrido-Maestu2017,
title = {Systematic loop-mediated isothermal amplification assays for rapid detection and characterization of Salmonella spp., Enteritidis and Typhimurium in food samples},
author = {Garrido-Maestu, A.; Fuciños, P.; Azinheiro, S.; Carvalho, J.; Prado, M.},
url = {http://www.sciencedirect.com/science/article/pii/S0956713517302542?via%3Dihub},
doi = {https://doi.org/10.1016/j.foodcont.2017.05.011},
year = {2017},
date = {2017-10-01},
journal = {Food Control},
volume = {80},
pages = {Pages 297-306},
abstract = {AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes. Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium},
keywords = {},
pubstate = {published},
tppubtype = {article}
}AbstractEuropean Authorities have made a great effort to decrease the incidence of salmonellosis, but yearly thousands of cases are still reported, being most of them associated with serovars Enteritidis and Typhimurium. In the current study a set of methods for fast detection of these pathogens was developed and evaluated. The methods were based on loop-mediated isothermal amplification due to its advantages. The methods targeted three genes, invA, safA and STM4497, and each one of them was evaluated independently so that they can be targeted directly or in a sequential mode: first screening for the genus Salmonella and secondly on typing those positive samples. In this process, the results were compared against qPCR. The methods were able to detect <10 cfu/25 g, making them suitable for official analyses, and food industry self-monitoring. Of most importance, the limit of detection, relative sensitivity, specificity and accuracy, positive and negative predictive values and the index kappa of concordance, were determined, being all higher than 97%. This demonstrates the reliability of the methods described in this study, which may be comparable with classical culture/serotyping of Salmonella but allowing a much faster response in case of positive results. Finally, a mathematical model was implemented to fit the data recorded by the qPCR thermocycler, allowing a more consistent determination of the parameters describing the qLAMP process, which may be easily implemented in other assays where accurate determination of Tt is needed for quantification purposes.
Keywords: qLAMP; qPCR; Salmonella spp.; S. Enteritidis; S. Typhimurium
Vial S.; Berrahal, Prado Wenger Y ; M ; J
Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates Journal Article
ACS Sensors , 2 (2) , pp. pp 251–256, 2017.
@article{Vial2017,
title = {Single-Step DNA Detection Assay Monitoring Dual-Color Light Scattering from Individual Metal Nanoparticle Aggregates},
author = {Vial, S.; Berrahal, Y.; Prado, M.; Wenger, J.},
url = {http://pubs.acs.org/doi/abs/10.1021/acssensors.6b00737},
doi = {10.1021/acssensors.6b00737},
year = {2017},
date = {2017-01-23},
journal = {ACS Sensors },
volume = {2 (2)},
pages = {pp 251–256},
abstract = {Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Efficiently detecting DNA sequences within a limited time is vital for disease screening and public health monitoring. This calls for a new method that combines high sensitivity, fast read-out time, and easy manipulation of the sample, avoiding the extensive steps of DNA amplification, purification, or grafting to a surface. Here, we introduce photon cross-correlation spectroscopy as a new method for specific DNA sensing with high sensitivity in a single-step homogeneous solution phase. Our approach is based on confocal dual-color illumination and detection of the scattering intensities from individual silver nanoparticles and gold nanorods. In the absence of the target DNA, the nanoparticles move independently and their respective scattering signals are uncorrelated. In the presence of the target DNA, the probe-functionalized gold and silver nanoparticles assemble via DNA hybridization with the target, giving rise to temporal coincidence between the signals scattered by each nanoparticle. The degree of coincidence accurately quantifies the amount of target DNA. To demonstrate the efficiency of our technique, we detect a specific DNA sequence of sesame, an allergenic food ingredient, for a range of concentration from 5 pM to 1.5 nM with a limit of detection of 1 pM. Our method is sensitive and specific enough to detect single nucleotide deletion and mismatch. With the dual-color scattering signals being much brighter than fluorescence-based analogs, the analysis is fast, quantitative, and simple to operate, making it valuable for biosensing applications.KEYWORDS: DNA detection; biosensing; dynamic light scattering; metal nanoparticles; optical scattering; plasmonics
-
2016
Prado M.; Espiña, Fernandez-Arguelles Diéguez Fuciños Vial Oliveira Reis Boehme B ; M T ; L ; P ; S ; J M ; R L ; K
Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends Book Chapter
Antimicrobial Food Packaging, pp. pp.183-202, Jorge Barros-Velazquez, 1st Edition, 2016, ISBN: 978-0-12-800723-5.
@inbook{Prado2016b,
title = {Detection of Foodborne Pathogens Using Nanoparticles. Advantages and Trends},
author = {Prado, M.; Espiña, B.; Fernandez-Arguelles, M. T.; Diéguez, L.; Fuciños, P.; Vial, S.; Oliveira, J. M.; Reis, R. L.; Boehme, K.},
url = {https://www.sciencedirect.com/science/book/9780128007235},
isbn = {978-0-12-800723-5},
year = {2016},
date = {2016-12-01},
booktitle = {Antimicrobial Food Packaging},
journal = {Academic Press, London},
pages = {pp.183-202},
publisher = {Jorge Barros-Velazquez},
edition = {1st Edition},
abstract = {Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.
},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}Due to their critical impact on public health, the detection of foodborne pathogens in food and water is an important issue for both, the food industry and control authorities. Fast and reliable analytical methods are needed in order to ensure the health of consumers, to easily determine whether a food product has been subjected to cross-contamination, and, simultaneously, to identify how and when this cross-contamination occurred in order to establish the proper corrective actions.Recent developments in nanotechnology are greatly impacting the advancement of some analytical techniques, including foodborne pathogen detection. Herein, the advantages and the recent applications of nanoparticles (NPs) in foodborne pathogen detection are reviewed. The most frequently used NPs in this context (gold nanoparticles, quantum dots, and magnetic nanoparticles) are described, and their applicability for food analysis is emphasized. Finally, this chapter provides an overview of the use of micro and nanofluidics, combined with nanoparticles, for food pathogen analysis, and the integration of nanomaterial-based sensors for pathogen detection in food packaging systems.Prado M.; Ortea, Vial Rivas Calo-Mata Barros-Velázquez I ; S ; J ; P ; J
Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens Journal Article
Critical Reviews in Food Science and Nutrition , 56 (Issue 15), pp. 2511-2542, 2016.
@article{Prado2016,
title = {Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens},
author = {Prado, M.; Ortea, I.; Vial, S.; Rivas, J.; Calo-Mata, P.; Barros-Velázquez, J.},
url = {http://www.tandfonline.com/doi/full/10.1080/10408398.2013.873767},
doi = {10.1080/10408398.2013.873767},
year = {2016},
date = {2016-11-17},
journal = {Critical Reviews in Food Science and Nutrition },
volume = {56},
number = {Issue 15},
pages = {2511-2542},
abstract = {Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles},
keywords = {},
pubstate = {published},
tppubtype = {article}
}Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.Keywords: Allergens, DNA based methods, proteomics, allergen detection, qPCR, biosensors, nanoparticles
-
2015
Sorry, no publications matched your criteria.
RESEARCH
DEPARTMENTS
RESEARCH GROUPS
HIGHLIGHTS
Paper published in Food Microbiology
Rapid and sensitive detection of viable Listeria monocytogenes in food products by a filtration-based protocol and qPCR. Alejandro Garrido-Maestu, , Sarah Azinheiro, Joana Carvalho, Marta Prado. Volume 73, August 2018, Pages 254–263.
Published a Research Article in Front. Sustain. Food Syst.
SarahAzinheiro,JoanaCarvalho,MartaPrado and AlejandroGarrido-Maestu. 21 February 2018.
Paper published in Microchemical Journal
Novel approach for accurate minute DNA quantification on microvolumetric solutions JoanaCarvalho,RenatoNegrinho,SarahAzinheiro,AlejandroGarrido-Maestu,JorgeBarros-Velázquez.MartaPrado.Volume 138, May 2018, Pages 540-549.
Paper published in Analytica chimica Acta.
JoanaCarvalho, GemaPuertas, JoãoGaspar, SarahAzinheiro, LorenaDiéguez, AlejandroGarrido-Maestu, ManuelVázquez,JorgeBarros-Velázquez,SusanaCardoso,MartaPrado.Volume 1020, 22 August 2018, Pages 30-40
NEWS & EVENTS
Author: Alejandro Garrido-Maestu, PabloFuciños, SarahAzinheiro, CarlaCarvalho, JoanaCarvalho, MartaPrado
Food Control 99 , pp. 79-83, 2019.
Book Chapter in collaboration with David Tomás Fornés, Lead Scientist in the Microbial and Molecular Analytics group at Nestlé Research Center (Lausanne, Switzerland). !!!!!! Foodborne Bacterial Pathogens
The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens In: Bridier A. (eds) Foodborne Bacterial Pathogens. Methods in Molecular Biology, vol 1918. Humana Press, New York, NY
New funded projects!
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PORT GRAPHE – Control of Port and Douro Wines authenticity using graphene DNA sensors project(Project Time Frame: June 2018 to June 2021)
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Nano BioSensor – Development of nanosensors to evaluate the microbiological quality of fruit-based products(Project Time Frame: July 2018 to June 2021)
GROUP LEADER
THE TEAM
Alejandro Garrido
Staff Researcher
Joana Guerreiro
Research Fellow
Monisha Elumalai
Research Fellow
Andrey Ipatov
Research Fellow
Jon Ashley
Research Fellow
Foteini Roumani
Research Fellow
Sarah Azinheiro
Research Laboratory Assistant
Joana Carvalho
Research Laboratory Assistant
Carlos Carpena
visitor from Ocupharm diagnostics S.L.
Shambhavi Yadav
Scientific Associate
FORMER GROUP MEMBERS
Karola Böhme (Research Fellow)
Master student ( February – September 2017)
University de Santiago de Compostela
Visitor (June 2017)
Sofia Granja Martins
Summer Student (July – September 2017)
María Leyva (Scientific Visitor)
Anna Toldrà Filella
Visitor from IRTA
Cristina Pastrana
visitor from Ocupharm diagnostics S.L.