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Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases

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Abstract

In this study, we describe a multiplex PCR method for the detection of five food-relevant virulence pathogenicity genes of intestinal pathogens. Five pairs of primers were designed based on nuc gene for Staphylococcus aureus, hlyA gene of Listeria monocytogenes, ipaH gene of Shigella flexneri, lysP gene of Yersinia enterocolitica and tpi gene of Clostridium difficile. Conditions were optimized to amplify fragments of those genes simultaneously in one PCR amplification. After developing and optimizing the multiplex PCR reaction system, the specificity and sensitivity of the multiple PCR assays were evaluated. The optimized program is also applied to retail meat for testing. The result indicated that when the annealing temperature was 54 °C and the primer concentrations of S. aureus, L. monocytogenes, S. flexneri, Y. enterocolitica and C. difficile are 10, 10, 5, 3 and 2 μM, the five strains could expand 484, 345, 204, 156, 88 bp of clear fragments, respectively. So was the multiple PCR in artificially contaminated beef produce. All cultures were cultured and separated by traditional methods. The multiplex PCR method offers a rapid, simple, and accurate identification of pathogens and could be used in food safety investigations, clinical diagnosis as well as for the surveillance of the spreading determinants of pathogens in epidemiological studies.

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Data Availability

The data used to support the findings of this study are included within the article.

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Funding

This work was supported by grants from the National Natural Science Foundation of China (81702103), Jiangsu Provincial Natural Science Foundation (BK20170252), Projects for Jiangsu Provincial Young Medical Talents (QNRC2016780).

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Author notes

  1. Ying Chen and Zixuan Wang authors contributed equally to this work.

Authors and Affiliations

  1. School of Medical Technology, Xuzhou Medical University, No. 209 Tongshan Road, Yunlong District, Xuzhou, 221004, China

    Ying Chen, Zixuan Wang, Qiaozhen Shi, Taotao Yu, Linyan Zhang & Huan Yang

  2. School of Food and Biological Engineering, Hefei University of Technology, Hefei, 230009, China

    Shengxiong Huang

Authors
  1. Ying Chen
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  2. Zixuan Wang
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  3. Qiaozhen Shi
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  4. Shengxiong Huang
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  5. Taotao Yu
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  6. Linyan Zhang
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  7. Huan Yang
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Contributions

YC and ZXW performed the statistical analyses and drafted the manuscript. QZS and SXH, TTY participated in data analysis. YC, ZXW, QZS, SXH, TTY, LYZ and HY conceived the study and participated in the design and coordination of the study. All authors read and approved the final manucript. YC and ZXW contributed equally to this work.

Corresponding author

Correspondence to Ying Chen.

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Chen, Y., Wang, Z., Shi, Q. et al. Multiplex PCR method for simultaneous detection of five pathogenic bacteria closely related to foodborne diseases. 3 Biotech 11, 219 (2021). https://doi.org/10.1007/s13205-021-02759-y

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