Novel Same-Day method for viable Salmonella Enteritidis detection in chicken meat combining phage amplification and LAMP

Salmonella enterica is a major foodborne pathogen worldwide. Poultry products, especially eggs and meat, are the main responsible for human salmonellosis cases. Culture-based methods require at least 3 days to detect Salmonella positive samples. To facilitate food chain processes and provide a rapid response to food outbreaks, a simple and rapid detection method is necessary. For this purpose, nucleic acid amplification-based techniques are a potential solution. Loop-Mediated isothermal AMPlification (LAMP) has emerged as an alternative to qPCR due to the simple equipment necessary to perform the analysis while allowing the detection of living cells when combined with bacteriophages. The aim of this work was to develop a same-day protocol based in the combination of LAMP and a Salmonella phage (vB-SenS_PVP-SE2) to detect viable Salmonella Enteritidis cells in chicken meat. Specific LAMP primers were designed to target the capsid and endolysin genes of Salmonella phage vB-SenS_PVP-SE2. Two different detection strategies were developed: real-time fluorescence; and colorimetric (naked-eye detection). The LAMP method developed could detect down to 0.2 fg/L of pure phage DNA and concentrations of viral particles in buffered peptone water (BPW) of 10 pfu/mL. After optimization in spiked chicken samples, a 3 h sample pre-enrichment diluted 1/10 in BPW before phage addition to the samples followed by a co-incubation (with phage) of 4 h was established. The proposed method could determine the presence of S. Enteritidis in less than 8 h including sample processing, DNA isolation and LAMP analysis with a LOD of 1.5 cfu/25g and a LOD of 6.6 cfu/25g, both by fluorescence and naked-eye observation. The results were in close concordance with the reference method for Salmonella spp., the ISO 6579-1:2017. The described method represents a promising alternative for the rapid detection of Salmonella in the food chain.info:eu-repo/semantics/publishedVersio

Specific detection of viable Salmonella Enteritidis by phage amplification combined with qPCR (PAA-qPCR) in spiked chicken meat samples

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/publishedVersio

Whole genome sequencing in the palm of your hand: how to implement a MinION Galaxy-based workflow in a food safety laboratory for rapid Salmonella spp. serotyping, virulence, and antimicrobial resistance gene identification

IntroductionWhole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment.MethodsHerein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced.ResultsAfter 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate.DiscussionThe implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates

Faster monitoring of the invasive alien species (IAS) Dreissena polymorpha in river basins through isothermal amplification

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 respectivelyThis work was supported by a partnership agreement project between the Confederación Hidrográfica del Guadalquivir and INL for the development of a system for early detection of zebra mussels through analysis of environmental DNA, and by project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. S. A. acknowledges the Portuguese funding institution FCT – Fundação para Ciência e Tecnologia for Ph.D. scholarship SFRH/BD/140396/2018S

Green synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment

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/publishedVersio

Interlaboratory validation of a multiplex qPCR method for the detection of listeria monocytogenes in a ready-to-eat seafood product

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 (<10CFU/25 g) with an overall agreement with the reference method, attending to the Cohen's k, of 0.97 that is interpreted as almost complete agreement.info:eu-repo/semantics/publishedVersio

Multiplex Detection of Salmonella spp., E. coli O157 and L. monocytogenes by qPCR Melt Curve Analysis in Spiked Infant Formula

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 &lt;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

Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces

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

Detection of foodborne pathogens by qPCR: A practical approach for food industry applications

Microbiological analysis of food is an integrated part of microbial safety management in the food chain. Monitoring and controlling foodborne pathogens are traditionally carried out by conventional microbiological methods based on culture-dependent approaches in control laboratories and private companies. However, polymerase chain reaction (PCR) has revolutionized microbiological analysis allowing detection of pathogenic microorganisms in food, without the necessity of classical isolation and identification. However, at present, PCR and quantitative polymerase chain reaction (qPCR) are essential analytical tools for researchers working in the field of foodborne pathogens. This manuscript reviews recently described qPCR methods applied for foodborne bacteria detection, serving as economical, safe, and reliable alternatives for application in the food industry and control laboratories. Multiplex qPCR, which allows the simultaneous detection of more than one pathogen in one single reaction, saving considerable effort, time, and money, is emphasized in the article