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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

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.

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.

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

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.