Determination and analysis of potential virulence of viable but nonculturable Vibrio parahaemolyticus

Objective To establish a real time RT-PCR method to detect VBNC Vibrio Parahaemolyticus and study the expression of virulence gene.Methods V.parahaemolyticus strain AS079 was induced into VBNC state by culturing in artificial seawater at 4 ℃.Real-time RT-PCR primers were designed for the toxR,pvuA and tdh2 gene.Various PCR protocol and primer concentration combinations were tested to optimize the PCR procedures for VBNC V.parahaemolyticus as well as virulence analysis.V.parahaemolyticus AS079 samples collected after various periods of incubation were analyzed using real-time RT-PCR method established in this study.Results The result indicated that the expression levels of virulence gene tdh2 and identification gene toxR decreased over incubation in artificial seawater.However,both genes were clearly detectable even after the bacteria had entered VBNC state,suggesting that the method could be used for detection and virulence analysis of V.parahaemolyticus under VBNC state.The sensitivity test showed that the detection limit for identification gene toxR was 48 cfu/ml;whereas evaluating the expression of virulence gene tdh2 required 4.8×102 cfu/ml at least.Meanwhile,no cross-reaction with other closely related food-borne pathogens had been found.Conclusion The real-time RT PCR method established in this study had advantages of fast detection,high specificity and sensitivity,and was suitable for detection and virulence analysis of VBNC V.parahaemolyticus.