Abstract:

Objective: To establish a specific method for the detection of Vibrio parahaemolyticus (VP) and its virulence
gene. Methods: Based on the sequences of toxR (transmembrane transcription activator) and tdh (thermostable direct
hemolysin) genes from VP, specific primers and TaqMan probes were designed and a duplex fluorenscencet real-time
PCR amplification system was established. The specificity and sensitivity of this method were evaluated. Moreover, the
distribution of virulence gene in import and export foods from Guangdong and Liaoning provinces was investigated by this
method. Results: The specificity tests showed that both toxR and tdh genes were amplified through the DNAs of standard
strain, ATCC33847, and 3 wild strains isolated from food-poisoning patients. However, no specific amplification curves
were observed for 31 other strains tested, including Vibrio alginolyticus and L. monocytogenes both from the genus Vibrio
and Enterobacteriaceae. The sensitivity results demonstrated an inverse linear relationship between VP concentration and
Ct values. The linear coefficients (R2) for toxR and tdh were 0.999 and 0.997 with an identical LOD of 3.6 × 102 CFU/mL,
respectively. Furthermore, the detection results revealed that toxR gene of all 37 foodborne VP wild strains in this study were
positive for toxR gene, but all of them were negative for tdh gene, indicating that the genomes of 37 wild strains contained
toxR gene, but did not contain tdh virulence gene. Conclusion: A rapid, sensitive and specific method suitable for the
simultaneous detection of species and virulences specific genes of VP in foods has been established in this study.

Key words: Vibrio parahaemolyticus, toxR gene, tdh gene, TaqMan based probe, duplex fluorescence real-time PCR, detection