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Microbial pathogens may be present in different types of foods, and hence the development of novel methods to assure consumers' safeness is of great interest. Molecular methods are known to provide sensitive and rapid results; however, they are typically targeted approaches. In recent years, the advent of non-targeted approaches based on next-generation sequencing (NGS) has emerged as a rational way to proceed. This technology allows for the detection of several pathogens simultaneously. Furthermore, with the same set of data, it is possible to characterize the microorganisms in terms of serotype, virulence, and/ or resistance genes, among other molecular features. In the current study, a novel method for the detection of Listeria monocytogenes based on the “quasimetagenomics” approach was developed. Different enrichment media and immunomagnetic separation (IMS) strategies were compared to determine the best approach in terms of L. monocytogenes sequences generated from smoked salmon samples. Finally, the data generated were analyzed with a user-friendly workflow that simultaneously provided the species identification, serotype, and antimicrobial resistance genes. The new method was thoroughly evaluated against a culture-based approach, using smoked salmon inoculated with L. monocytogenes as the matrix of choice. The sequencing method reached a very low limit of detection (LOD50, 1.2 CFU/ 25 g) along with high diagnostic sensitivity and specificity (100%), and a perfect correlation with the culture-based method (Cohen's k = 1.00). Overall, the proposed method overcomes all the major limitations reported for the implementation of NGS as a routine food testing technology and paves the way for future developments taking its advantage into consideration.

Abstract Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectively.

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%.

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.

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.

The authors would like to make the following correction to the published paper [...]

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.

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 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.

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.

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.