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Development and evaluation of a dual priming oligonucleotide system-based multiplex PCR assay for simultaneous detection of six foodborne pathogens

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Abstract

In this study, a multiplex PCR assay with dual priming oligonucleotide system (DPO system-based mPCR) was developed for the simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Shigella spp., Staphylococcus (S.) aureus, Campylobacter (C.) jejuni and Yersinia (Y.) enterocolitica from food. Pathogen-specific DPO systems were designed targeting Salmonella spp. fimY gene, L. monocytogenes iap gene, Shigella spp. ipaH gene, S. aureus 442 gene, C. jejuni gyrA gene and Y. enterocolitica 16 s–23 s rRNA gene, respectively. Our optimized DPO system-based mPCR assay allowed a wide range of annealing temperature from 48 to 68 °C to efficiently amplify multi-genes followed by a nearly identical pattern with an analytical detection limit of 102–103 CFU/mL (g) for the simultaneous detection of the six target bacteria in pure cultures or artificially contaminated food matrixes. Applying the DPO system-based mPCR assay to 238 target and 83 nontarget bacterial strains revealed that only target bacterial strains were positive in this assay, indicating a high specificity. Moreover, the DPO system-based mPCR assay showed a potential diagnostic capability evaluated by testing 2419 samples collected from food, clinical and environmental sources. The DPO system-based mPCR assay may provide a useful tool for the detection of these six foodborne pathogens in laboratory diagnosis.

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Acknowledgments

This work was supported by the Projects of AQSIQ (Grant 2013IK051), Special Projects of Quality Inspection Nonprofit Industry (Grant 201310126) and Natural Science Foundation of Heilongjiang Province (Grant C201349).

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Authors and Affiliations

  1. College of Veterinary Medicine, Northeast Agricultural University, Mu Cai Street 59, Xiang Fang District, Harbin, China

    Yi-Gang Xu, Bo Sun, Hai-Yuan Zhao, Yan-Ping Jiang, Li Wang, Xin-Yuan Qiao, Yi-Jing Li & Li-Jie Tang

  2. Technology Center, Heilongjiang Entry-Exit Inspection and Quarantine Bureau, Harbin, China

    Zhong-Mei Liu

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  1. Yi-Gang Xu
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  2. Bo Sun
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  3. Hai-Yuan Zhao
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Correspondence to Li-Jie Tang.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Xu, YG., Sun, B., Zhao, HY. et al. Development and evaluation of a dual priming oligonucleotide system-based multiplex PCR assay for simultaneous detection of six foodborne pathogens. Eur Food Res Technol 243, 555–563 (2017). https://doi.org/10.1007/s00217-016-2765-2

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  • DOI: https://doi.org/10.1007/s00217-016-2765-2

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