Establishment of a quadruple PCR method for detecting pathogenic genes in Vibrio parahaemolyticus

Abstract:

[Objective] This study aimed at establishment of a rapid specific quadruple PCR assay for detecting Vibrio parahaemolyticus and its virulence genes. [Methods] Four pairs of primers were designed based on toxR, tdh, trh and tlh genes sequences of V. parahaemolyticus. Optimized the concentration of four pairs of primers and the annealing temperature to obtain the best ratio of primer and amplification conditions, the rapid quadruple PCR detection of pathogenic V. parahaemolyticus was established. The specificity and sensitivity of the quadruple PCR method were also evaluated. [Results] The expected sizes of amplification bands were 115 bp, 244 bp, 418 bp and 759 bp for toxR, tdh, trh and tlh, respectively. The highly specificity of this method was evaluated by using 74 V. parahaemolyticus strains and 37 non-targeted bacteria. The detection limits of the multiplex PCR for DNA was 50 μg/L. Detection sensitivity of V. parahaemolyticus in pure culture condition was 6.7×103 CFU/mL. Four targeted fragments were detected in artificially contaminated seafood with 1.36 CFU/g V. parahaemolyticus after 6 h enrichment. [Conclusion] The multiplex-PCR method can simultaneously detect V. parahaemolyticus with toxR, tdh, trh and/or tlh. It is of certain significance to carry out the detection of pathogenic V. parahaemolyticus.

Key words:Vibrio parahaemolyticus, Quadruple PCR, toxR gene, tdh gene, trh gene, tlh gene