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Background: Ready-to-eat products, such as leafy greens, must be carefully controlled as they are directly consumed without any treatment to reduce the presence of potential pathogens. Food industries, especially those that process products with short shelf-life, demand rapid detection of foodborne pathogens such as Shiga Toxinproducing Escherichia coli (STEC). In this sense, molecular methods can fulfill both requirements of turnaround time and consumer safety. The most popular rapid methods are those based on real-time PCR (qPCR) however, vegetables contain inhibitory compounds that may inhibit the amplification reaction thus, there is a need for novel sample preparation protocols. Results: In the current study, a low-cost sample treatment based on sequential filtration steps was developed. This protocol was combined with covalent organic frameworks (COFs), and compared against a chelating resin, to evaluate their performance by multiplex qPCR targeting the major virulence genes of STEC, namely stx1, stx2, and eae, along with the rfbE for the specific identification of serogroup O157 due to its particularly high incidence, and an Internal Amplification Control to assess reaction inhibition. The optimized sample treatment effectively removed vegetable qPCR inhibitory compounds, and it was possible to detect STEC in spiked ready-toeat salad samples in one working day, roughly 5 h, with an LOD50 of 8.7 CFU/25 g with high diagnostic sensitivity and specificity. The method was also assessed in samples with cold-stressed bacteria with good results, further demonstrating its applicability. Significance: It was demonstrated for the first time that COFs are suitable for DNA extraction and purification. In addition to this, due to the tunable nature of these materials, it is envisioned that future modifications in terms of pore size or combination with magnetic materials, will allow to further improve their performance. In addition to this, the rapid and low-cost sample treatment protocol developed demonstrated suitable for the rapid screening of STEC vegetable samples. published

6 pages, 4 tables, 1 figure.-- Under a Creative Commons license, Listeria monocytogenes is a major foodborne pathogen which mainly infects susceptible individuals through the consumption of contaminated foods. To this end, ready-to-eat (RTE) food products are of particular concern as this microorganism is widely distributed, can survive, and even grow, under adverse conditions, and thus must be carefully controlled. In the present study, an interlaboratory ring trial was organized to evaluate an open formula qPCR-based method for the detection of L. monocytogenes. The molecular method was evaluated on a novel RTE seafood product, developed in the framework of a European project, the SEAFOODAGE (EAPA_758/2018). Six laboratories located in Spain and Portugal participated in the study, and the results obtained indicated that this new method presented high diagnostic sensitivity (100%) reaching a low limit of detection (, This work was financially supported by the Seafood Age project, which was co-financed by the Interreg Atlantic Area Program (EAPA_758/2018) though the European Development Fund (ERDF). Mrs. Sarah Azinheiro was financed by a Ph.D. grant from the Fundação para a Ciência e a Tecnologia (SFRH/BD/140396/2018). Dr. Alejandro Garrido-Maestu and Luís D. R. Melo acknowledge funding from the Fundação para a Ciência e Tecnologia through the Scientific Employment Stimulus Program (2021.02810. CEECIND and 2021.00221. CEECIND, respectively). This study was supported by the Fundação para a Ciência e a Tecnologia (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit

Foodborne pathogens are still a significant source of morbidity and mortality worldwide. In addition to this the current methodologies to track these microorganisms cannot cope with the current intensive production systems, thus novel methods are of outmost importance. DNA-based methods have already demonstrated suitable to address this issue, but most of them are targeted methods such as real-time PCR (qPCR), meaning that one will only find what is looking for, thus taking the risk of missing relevant pathogens in a given sample. To overcome this limitation we have developed an easy-to-implement methodology which enables the detection of several pathogens simultaneously by using long-read Next Generation Sequencing (NGS) with MinION. The method was named “semi-targeted” due to the combination of a non-targeted detection method, NGS, with the usage of selective media in order to partially eliminate non-pathogenic interfering bacteria. To this end, we included an enrichment step for the recovery of different pathogens, namely Salmonella Enteritidis and Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7, after DNA extraction and library preparation, the samples were analyzed with MinION implementing the low-cost Flongle Flow Cells. The methodology was successfully evaluated in spiked milk samples with an excellent agreement with the results obtained by qPCR and culture-based methods. The method can provide accurate results after only 2 h of sequencing. Sample multiplexing, along with the lower cost of the Flongle Flow Cells and the reduced price of the MinION platform, make the assay cost-effective that is of importance for the food industry. Starting the method with a classical microbiological approach, the enrichment, the method is easy to implement in testing laboratories, it provides flexibility in terms of potential pathogens to be detected, and the positive results can be easily confirmed following culture-based, or other type, of confirmation procedures.

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.

Miniaturization of DNA-based techniques can bring interesting advantages for food analysis, such as portability of complex analytical procedures. In the olive oil industry, miniaturization can be particularly interesting for authenticity and traceability applications, through in situ control of raw materials before production and/or the final products. However, variety identification is challenging, and implementation on miniaturized settings must be carefully evaluated, starting from the selected analytical approach. In this work, SSR- and SNP-based genotyping strategies were investigated for the identification and differentiation of two olive varieties from the Northwest of Spain. For the selected SNPs two genotyping methods were tested: real-time allele-specific PCR and high resolution melting analysis. These methods were compared and evaluated regarding their potential for integration in a microfluidic device. Both SNP-based methods proved to be successful for identification of the selected varieties, however real-time allele-specific PCR was the one that achieved the best results when analyzing mixtures, allowing the identification of both monovarietal samples and mixtures of the varieties tested with up to 25%.

Lignin particles (LPs) have gained prominence due to their biodegradability and bioactive properties. LP production at nano and micro scale produced from organosolv lignin and the understanding of size's effect on their properties is unexplored. This work aimed to produce and characterize lignin nanoparticles and microparticles using a green synthesis process, based on ethanol-solubilized lignin and water. Spherical shape LPs, with a mean size of 75 nm and 215 nm and with a low polydispersity were produced, as confirmed by transmission electron microscopy and dynamic light scattering. LPs thermal stability improved over raw lignin, and the chemical structure of lignin was not affected by the production method. The antimicrobial tests proved that LPs presented a bacteriostatic effect on Escherichiacoli and Salmonella enterica. Regarding the antioxidant potential, LPs had a good antioxidant activity that increased with the reaction time and LPs concentration. LPs also presented an antioxidant effect against intracellular ROS, reducing the intracellular ROS levels significantly. Furthermore, the LPs showed a low cytotoxic effect in Caco-2 cell line. These results showed that LPs at different scales (nano and micro) present biological properties and are safe to be used in different high value industrial sectors, such as biomedical, pharmaceutical and food., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit, BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, and MICRODIGEST project (grant agreement 037716) co-funded by FCT and ERDF through COMPETE2020., info:eu-repo/semantics/publishedVersion

Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.

Escherichia coli O157 continues to be the most prevalent serotype among the Shiga toxin-producing E. coli infection cases confirmed in Europe. The reference methodology to detect this pathogen is lengthy and time consuming, thus we sought to develop a novel method that has low instrumentation requirement, and allowed naked-eye detection. Isothermal amplification of bacterial DNA was performed by Recombinase Polymerase Amplification, and the addition of SYBR Green I (RPA-SG), which allowed the visualization of results with naked-eye under a UV lamp. The results obtained in spiked ground meat samples by RPA-SG compared favorably to qPCR (relative sensitivity, specificity and accuracy higher than 90%, and Cohen's k of 0.81), with a limit of detection of 19 cfu/25 g. The novel methodology outperformed a culture-based approach, where none of the typical colonies were confirmed as O157 due to high concentration of interfering microorganisms. These results were obtained in one working day (same-day detection), having an average time to completion of about 5 h, including enrichment, DNA extraction, amplification and detection.

Purification and concentration of DNA is a critical step on DNA-based analysis, which should ensure efficient DNA isolation and effective removal of contaminants that may interfere with downstream DNA amplification. Complexity of samples, minute content of target analyte, or high DNA fragmentation greatly entangles the success of this step. To overcome this issue, we designed and fabricated a novel miniaturized disposable device for a highly efficient DNA purification. The microfluidic device showed binding efficiency and elution yield of 90.1% and 86.7%, respectively. Moreover, the effect of DNA fragmentation, a parameter that has not been previously addressed, showed a great impact in the recovery step. The microfluidic system integrated micropillars with chitosan being used as the solid-phase for a pH-dependent DNA capture and release. We have showed the potential of the device in the successful purification of environmental DNA (eDNA) from river water samples contaminated with Dreissena polymorpha, an invasive alien species responsible for unquestionable economic and environmental consequences in river water basins. Additionally, the device was also able to concentrate the DNA extract from highly diluted samples, showing promising results for the early detection of such invasive species, which may allow prompt measures for a more efficient control in affected areas. Suitability for integration with downstream DNA analysis was also demonstrated through qPCR analysis of the samples purified with the microfluidic device, allowing detection of the target species even if highly diluted.

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/&micro, L 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.

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.

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.

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.

Nowadays, the most widely accepted rapid methods in food microbiology rely on nucleic acid amplification such as PCR/real-time PCR. A major claimed limitation of these methods is their incapacity to differentiate among viable and non-viable microorganisms. In the present study we report the development of a novel multiplex loop-mediated isothermal amplification method which, by combining immunomagnetic separation, to concentrate and purify the bacteria, along with propidium monoazide to block the amplification of DNA from non-viable microorganisms. The method allowed to specifically detect viable Listeria monocytogenes present in milk products. We designed an internal amplification control to rule out false negative results due to reaction inhibition, which is differentiated from L. monocytogenes by a simple melt curve analysis. Overall, the methodology provided results higher than 95% in terms of sensitivity, specificity and accuracy, as well as a Cohen's k of 0.97, reaching a limit of detection of 2.7 cfu/25 g. In samples inoculated with up to 106-107 cfu of dead microorganisms, the method demonstrated capable of effectively eliminating undesired amplification.

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.

European 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

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/L of pure virus DNA and a concentration of viral particles of 103pfu/mL. After a short bacterial recovery step, the addition of bacteriophages to spiked chicken samples indicated that 8cfu/25g could be detected within 10h, 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., This work was supported by the project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by NortePortugal Regional Operational Programme(NORTE2020), andby the “NanoBioSensor: Desenvolvimento de nanosensores para avaliação da qualidade microbiológica de produtos à base de fruta” (POCI-010247-FEDER-033925), supported bytheOperational ThematicProgram for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

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. PRACTICAL APPLICATION: The methodology described in this study significantly reduces the detection time of L. monocytogenes, when compared with culture-based methods, and it requires fewer steps than other molecular methods, making it a reliable and more convenient method for routine testing. Finally, the evaluation of the methodology in spiked food samples, confirms its reliability.

Dairy products have been implicated in foodborne infections caused by different bacterial pathogens. Among them, Listeria monocytogenes is of particular concern due to its ubiquity, resistance to sanitation processes and high mortality rates resulting from infection. These issues make the development of novel methods for the rapid detection of this bacterium of high interest. The evaluation of a novel multiplex real-time Recombinase Polymerase Amplification method including an internal amplification control is reported in the present work. The method performance was compared to that of the European reference method (ISO 11290-1) for the detection of the species in samples from 40 commercial products, including 14 UHT milk samples, 16 hard cheese samples, 6 infant dairy preparation samples and 4 fresh cheese samples. A limit of detection below 10 cfu/25 g or mL sample was achieved, and values higher than 90% were obtained for relative sensitivity, specificity, accuracy, positive and negative predictive values and the index (kappa) of concordance. Analysis was achieved within one working day, compared to the six days required using the ISO method. Moreover, slight modification of the ISO 11290-1 method to include secondary enrichment in half Fraser broth resulted in the confirmation of all positive samples.

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.

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.

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.

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.

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.

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.

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.