Detection and differentiation of Vibrio parahaemolyticus by multiplexed real-time PCR

• Published in: Canadian journal of microbiology Vol. 64; no. 11; pp. 809 - 815
• Main Authors: Xu, Deshun, Ji, Lei, Wu, Xiaofang, Yan, Wei, Chen, Liping
• Format: Journal Article
• Language: English
• Published: Canada NRC Research Press 01.11.2018; Canadian Science Publishing NRC Research Press
• Subjects: Animals; Assaying; avirulent strains; bacteria; Bacterial Proteins - genetics; Bacterial Toxins - genetics; cross reaction; Cross-reactivity; detection limit; Detection limits; Differentiation; disease incidence; DNA Primers - chemistry; DNA, Bacterial - genetics; Dosage; Gastroenteritis; genes; Hemolysin Proteins - genetics; Humans; Microbiology; Multiplex Polymerase Chain Reaction - methods; multiplex real-time PCR; Multiplexing; nucleic acid annealing; Ostreidae - microbiology; Outbreaks; Oysters; pathogen; Pathogens; pathogène; PCR multiplex en temps réel; quantitative polymerase chain reaction; Real time; Seafood; sensibilité; sensitivity; Sensitivity analysis; Sensitivity and Specificity; specificity; spécificité; Strains (organisms); temperature; Vibrio Infections - diagnosis; Vibrio parahaemolyticus; Vibrio parahaemolyticus - classification; Vibrio parahaemolyticus - genetics; Virulence; Waterborne diseases; PCR multiplex en temps réel; sensibilité; multiplex real-time PCR; Vibrio parahaemolyticus; specificity; pathogen; pathogène; sensitivity; spécificité
• ISSN: 0008-4166; 1480-3275; 1480-3275
• DOI: 10.1139/cjm-2018-0083

Vibrio parahaemolyticus is a common and important pathogen that causes human gastroenteritis worldwide. A rapid, sensitive, and specific assay is urgently required for detection and differentiation of V. parahaemolyticus strains. We designed three sets of primers and probes using groEL and two virulence genes (tdh and trh) from V. parahaemolyticus, and developed a multiplex real-time PCR protocol. The sensitivity and specificity of the multiplex assay was evaluated by environmental and clinical specimens of V. parahaemolyticus. The multiplex PCR response system and annealing temperature were optimized. The detection limits of the multiplex real-time PCR were 10 4 and 10 5 CFU/mL (or CFU/g) in pure cultures and spiked oysters, respectively. The multiplex real-time PCR specifically detected and differentiated V. parahaemolyticus from 35 Vibrio strains and 11 other bacterial strains. Moreover, this method can detect and distinguish virulent from nonvirulent strains, with no cross-reactivity observed in the bacteria tested. This newly established multiplex real-time PCR assay offers rapid, specific, and reliable detection of the total and pathogenic V. parahaemolyticus strains, which is very useful during outbreaks and sporadic cases caused by V. parahaemolyticus infection.