AUTHOR=Niu Ben , Hong Bin , Zhang Zhaohuan , Mu Lili , Malakar Pradeep K. , Liu Haiquan , Pan Yingjie , Zhao Yong 

TITLE=A Novel qPCR Method for Simultaneous Detection and Quantification of Viable Pathogenic and Non-pathogenic Vibrio parahaemolyticus (tlh+, tdh+, and ureR+)

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01747

DOI=10.3389/fmicb.2018.01747

ISSN=1664-302X

ABSTRACT=<p>Pathogenic and non-pathogenic <italic>Vibrio parahaemolyticus</italic> strains were simultaneously detected and quantified using a novel viable multiplex real-time PCR (novel qPCR). We used a new PCR primer and probe, <italic>ure</italic>R, as a surrogate for detection of the toxin <italic>trh</italic> gene as the primer was better at identifying variant <italic>V. parahaemolyticus trh</italic> strains. The specificity of all primers and probes used in this study were validated on three standard strains of <italic>V. parahaemolyticus</italic>, 42 clinical strains, 12 wild strains, 4 strains of <italic>Vibrio</italic> 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. Pathogenic and non-pathogenic strains were quantified using a two-reaction tube method where the <italic>tlh</italic>, <italic>tdh</italic>, and <italic>ure</italic>R genes were amplified. Additionally, standard curves with a 7-log dynamic range were generated for quantifying viable <italic>V. parahaemolyticus</italic> and the amplification efficiencies were 108.68, 105.17, and 115.61% for <italic>tlh<sup>+</sup></italic>, <italic>tdh<sup>+</sup></italic>, and <italic>ure</italic>R<italic><sup>+</sup></italic>. This novel qPCR accurately monitored <italic>V. parahaemolyticus</italic> contamination rates in shrimps (<italic>Penaeus vannamei</italic>) and clams (<italic>Ruditapes philippinarum</italic>) sampled from retail stores located in a major district in Shanghai. In conclusion, our assay can prioritize the detection and quantification of viable pathogenic <italic>V. parahaemolyticus</italic> and can prove to be a more effective tool for reducing infection risks from consumption of seafood in Shanghai.</p>