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Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

Abstract

A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 108 CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 101–102 CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes, respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

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Acknowledgments

This research was supported by the National Natural Science Foundation of China (31271870), the project of Science and Technology Commission of Shanghai Municipality (14DZ1205100, 14320502100, 12391901300), Key Project of Shanghai Agriculture Prosperity through Science and Technology (Grant: 2014, 3–5; 2015, 4–8), and Cross-discipline project (B5201120040). Pradeep Malakar was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK.

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The authors declare that they have no conflict of interests.

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Correspondence to Yong Zhao.

Additional information

Zhaohuan Zhang and Haiquan Liu contributed equally to this work.

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Zhang, Z., Liu, H., Lou, Y. et al. Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp. Appl Microbiol Biotechnol 99, 6451–6462 (2015). https://doi.org/10.1007/s00253-015-6715-x

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Keywords

  • TaqMan-based multiplex real-time PCR
  • Propidium monoazide
  • Viable foodborne pathogens
  • Vibrio parahaemolyticus
  • Listeria monocytogenes
  • Raw shrimp