We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5–25 viral RNA copies per μl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/μl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/μl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

This study was aimed at developing a one-tube multiplex real-time PCR system for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Vibrio parahaemolyticus and Shigella spp.) in food samples. The newly developed multiplex real-time PCR was performed using fluorescent dye EvaGreen and melting curve analysis for pathogen discrimination. The specificity of the PCR method was evaluated by testing with DNA extracted from different strains. The detection limits of genomic DNA per reaction for the five pathogens ranged from 0.39 pg (Shigella spp.) to 40 pg (Listeria monocytogenes). Sensitivity and specificity of this multiplex real-time PCR were further confirmed using artificially and naturally contaminated samples. The newly developed multiplex real-time PCR was useful for rapid, specific and cost-effective detection of the five target pathogens in contaminated food samples. PRACTICAL APPLICATIONSThe newly developed multiplex real-time PCR assay could detect five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Vibrio parahaemolyticus and Shigella spp.) in food samples in a single reaction tube. This method is simple, cost-effective, specific, sensitive and is capable of high-throughput detection for five foodborne pathogens simultaneously. Therefore, it has a potential application as a useful tool in food testing laboratories for quick identification of foodborne pathogens.

Salmonella , a major foodborne pathogen, causes severe gastrointestinal disease in people and animals worldwide. Plasmid-borne mcr-1 , which confers colistin resistance in Salmonella, has significant epidemiological interest for public health safety. Here, we report the first evidence of mcr-1 -mediated colistin resistance in one multidrug-resistant strain，namely 16062 in this study, from 355 Salmonella isolates collected for Jiaxing foodborne pathogen monitoring in Zhejiang Province in 2015–2019. In addition to colistin, 16062 displayed multidrug resistance to various antimicrobials (β-lactams, quinolone, sulfonamide, florfenicol, ampicillin, streptomycin, nalidixic acid, aminoglycoside, and trimethoprim-sulfamethox). The mcr-1 -carrying IncX4 plasmid (p16062-MCR) in this study shares a conserved structure with other mcr -IncX4 plasmids. We found that other antimicrobial-resistance genes ( aac(6')-Ib-cr , aadA1 , aadA2 , aph(3')-Ia , oqxA , oqxB , sul1 , and cmlA1 ) are located on p16062-cmlA, an atypical IncHI2 plasmid, in isolate 16062. This is the first identification of transferable colistin resistance in foodborne Salmonella isolate collected in Jiaxing city, the 5-year monitoring of which revealed limited dissemination. By determining the genetic features of the plasmid vehicle, the characteristics of transferable mcr genes circulating in isolates from Jiaxing are now clearer.

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