关键词: 副溶血弧菌；多重实时聚合酶链式反应；快速检测；毒力基因

Abstract: In this study, in order to establish a triplex real-time polymerase chain reaction (real-time-PCR) method based on a Taqman probe for the rapid detection of the virulence genes of Vibrio parahaemolyticus, primers and probes were designed targeting V. parahaemolyticus-specific genes and its main virulence genes tlh, tdh and ureR, and the reaction conditions were optimized. Ten strains of V. parahaemolyticus and 22 non-V. parahaemolyticus strains were used to verify the specificity of the designed primers and probes. The results showed that the primers and probes presented high specificity. The optimized primer concentrations were 0.15, 0.15 and 0.80 μmol/L for tdh, tlh and ureR, respectively, and the optimized probe concentrations were 0.50 μmol/L for both HEX and FAM. Even at a high concentration of background bacteria, this method exhibited a good linear relationship between DNA concentration and Ct value, and the lowest detection limit was 1.8 × 102 copies/mL. When the pre-denaturation time was set to 30 min, the amplification result using the intact bacterial cell suspension as the template was comparable to that using genomic DNA (gDNA) (ΔCt < 1). The triplex real-time PCR method can not only allow for rapid and quantitative detection of V. parahaemolyticus, but also effectively distinguish between pathogenic and non-pathogenic V. parahaemolyticus, thus providing a rapid, sensitive and accurate method for risk assessment of pathogenic V. parahaemolyticus.

Key words: Vibrio parahaemolyticus; multiplex real-time polymerase chain reaction; rapid detection; virulence genes