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A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk

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Abstract

Escherichia coli and Shigella spp. are two major bacterial contaminants in raw milk. Effective screening of the two microbes before starting the production process is a critical step for the quality and safety guarantee of the resulting dairy products. This study reported a rapid and simple realtime PCR assay using a ubiquitous primer and probe set targeting the tuf gene for the detection of E. coli and Shigella spp. An internal amplification control (IAC) was also comprised to indicate false-negative results. The duplex realtime PCR assay showed a high efficiency above 96 % and a detection limit <10 cfu per PCR. When artificially contaminated raw milk samples were further evaluated, the assay performed equally as well as the traditionally cultural-based method, and facilitated quantitative detection of the two microbes in the range from ~102 to ~106 cfu mL−1 raw milk. The detection limit was ~102 cfu mL−1 raw milk for either or a mixture of the two strains without pre-enrichment step, and could be <10 cfu per 10 mL of raw milk if a pre-enrichment step was added. Considering the detection effectiveness, time-consumption saving, and economical efficiency, the present duplex realtime PCR assay has a great potential in the application in raw milk for assessing their microbiological quality and safety in relation to E. coli and Shigella spp.

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Acknowledgments

The authors gratefully acknowledge the financial assistance provided by the Open Project Program of State Key Laboratory of Dairy Biotechnology, Bright Dairy & Food Co. Ltd. (No. SKLDB2012-009).

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Authors and Affiliations

  1. State Key Laboratory of Dairy Biotechnology, Bright Dairy & Food Co. Ltd., No. 1518 West Jiangchang Road, Shanghai, 200436, China

    Yuexia Wang & Wanyi Chen

  2. College of Life Sciences, Henan Agricultural University, 63 Nongye Rd., Zhengzhou, 450002, Henan, China

    Yuexia Wang, Pengfei Zhao, Huanling Zhang & Xiaoyu Su

  3. College of Food Science & Technology, Henan Agricultural University, 63 Nongye Rd., Zhengzhou, 450002, Henan, China

    Biao Suo

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  1. Yuexia Wang
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  2. Pengfei Zhao
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  3. Huanling Zhang
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  4. Wanyi Chen
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  5. Xiaoyu Su
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  6. Biao Suo
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Corresponding author

Correspondence to Biao Suo.

Additional information

Y. Wang and P. Zhao contributed equally to this work.

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Wang, Y., Zhao, P., Zhang, H. et al. A simple and rapid realtime PCR assay for the detection of Shigella and Escherichia coli species in raw milk. J. Verbr. Lebensm. 8, 313–319 (2013). https://doi.org/10.1007/s00003-013-0837-9

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  • DOI: https://doi.org/10.1007/s00003-013-0837-9

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