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Front. Microbiol. | doi: 10.3389/fmicb.2018.01747

A novel qPCR method for simultaneous detection and quantification of viable pathogenic and non-pathogenic Vibrio parahaemolyticus (tlh+, tdh+, ureR+)

 Ben Niu1, Lili Mu1,  Zhaohuan Zhang1,  Bin Hong1,  Haiquan Liu1, Yingjie Pan1 and  Yong Zhao1*
  • 1Shanghai Ocean University, China

ABSTRACT
A novel viable multiplex real-time PCR (novel qPCR) was developed for the simultaneous detection and quantification of pathogenic and non-pathogenic Vibrio parahaemolyticus (V. parahaemolyticus). In this study, the new primer and probe of ureR was used as a surrogate for detection of trh to circumvent the variant trh strains, and the specificity of all primers and probes were tested by 3 standard strains of V. parahaemolyticus,42 clinical strains, 12 wild strains, 4 strains of vibrio spp, and 4 strains of other bacteria. Then, propidium monoazide (PMA) was applied to inhibit DNA of dead cell, and the results of PMA optimized treatments were 15 µM concentration, 5 min incubation periods, 15 min light exposure periods and 30 RPM rotational speed, which resulted in time and cost savings. In addition, two reaction tubes were innovatively proposed to amplify tlh, tdh and ureR respectively, overcoming the complex logical relation of pathogenic and non-pathogenic V. parahaemolyticus. Furthermore, optimal standard curves with a 7-log dynamic range were generated for simultaneously quantifying viable V. parahaemolyticus, and its amplification efficiencies were 108.68, 105.17 and 115.61 % for tlh+, tdh+ and ureR+ respectively. At last, this new assay has been successfully employed to monitor the V. parahaemolyticus contamination rate of shrimp (Penaeus vannamei) and clam (ruditapes philippinarum) from the retail store of major district in Shanghai. In conclusion, this novel qPCR was a powerful method for detection and quantification of viable pathogenic and non-pathogenic V. parahaemolyticus in natural sample, which could support an effective and rapid tool for the authority to monitor the contamination of viable V. parahaemolyticus.

Keywords: Novel qPCR, PMA, Pathogenic and non-pathogenic V. parahaemolyticus, Rapid detection technique, shrimp, clam

Received: 09 May 2018; Accepted: 12 Jul 2018.

Edited by:

Qingli Dong, University of Shanghai for Science and Technology, China

Reviewed by:

Learn-Han Lee, Novel Bacteria and Drug Discovery Research Group (NBDD), Monash University, Malaysia, Malaysia
Dapeng Wang, Shanghai Jiao Tong University, China  

Copyright: © 2018 Niu, Mu, Zhang, Hong, Liu, Pan and Zhao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Yong Zhao, Shanghai Ocean University, Shanghai, China, yzhao@shou.edu.cn