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

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

doi:10.3389/fmicb.2018.01747

Ben Niu, Pradeep K Malakar + Show 6 more

Open Access

https://doi.org/10.3389/fmicb.2018.01747

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Journal: Frontiers in microbiology	Publication Date: Aug 2, 2018
Citations: 19	License type: CC BY 4.0

Affiliation: Shanghai Ocean University

Abstract

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

Highlights

Vibrio parahaemolyticus is a gram-negative and halophilic pathogen which was identified in the 1950s (Shinoda, 2011)
The V. parahaemolyticus virulence factors contributing to disease include adhesin, thermostable direct hemolysin (TDH), TDH related hemolysin (TRH), 2 type III secretion systems (T3SS1 and T3SS2) and 2 type VI secretion systems (T6SS1 and T6SS) (Ceccarelli et al, 2013; Wang R. et al, 2015)
V. parahaemolyticus strains were grown in 9 mL tryptic soy broth (TSB; Beijing Land Bridge Technology Company Ltd., Beijing, China) with 3% (w/w) sodium chloride (NaCl) and incubated at 37◦C for 16–18 h, and other strains were grown in 9 mL TSB at 37◦C for 18–20 h to get approximately 9 Log CFU/g

Summary

Introduction

Vibrio parahaemolyticus is a gram-negative and halophilic pathogen which was identified in the 1950s (Shinoda, 2011) This bacterium is widely distributed in the marine and estuarine environment (Bej et al, 1999) and is a major food borne pathogen in China and South East Asia countries (Su and Liu, 2007; Zhang et al, 2015a). It is the common cause of gastrointestinal disease from consumption of raw or lightly cooked seafood (Letchumanan et al, 2015; Eschbach et al, 2017). The ureR gene cluster is immediately upstream of trh and using this cluster can circumvent challenges associated with trh variation (Suthienkul et al, 1995; Nilsson and Turner, 2016)

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