Detection of Vibrio parahaemolyticus virulence gene by cross primer thermostatic amplification technology

Abstract

Objective To establish a CPA-nucleic acid test strip method for rapid detection of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) and heat-labile hemolysin (TLH). Methods Primers and probes were designed according to the sequence of TDH and TLH virulence genes of Vibrio parahaemolyticus, The optimal reaction temperature and time were established by optimizing the reaction system. The CPA method was compared with the fluorescence quantitative PCR. Results The optimal temperature and time for the CPA-nucleic acid test strip method established in this study were 60 ℃ and 40 min, which were highly specific to the TDH and TLH virulence genes of Vibrio parahaemolyticus. The TLH And TDH detection limit of 1.8 cfu/mL and 16.0 cfu/mL; consistent with the method of fluorescence quantitative PCR, and has good stability. The positive rates of TLH and TDH genes were 100% (464/464) and 72.6% (337/464) in 464 isolates, respectively. The positive rate of TDH virulence genes in isolates of aquatic products was 7.81 % (10/128). The positive rate of TDH virulence gene in clinical isolates was 97.32% (327/336). Conclusions The CPA-nucleic acid test strip method for detection of Vibrio parahaemolyticus TDH and TLH genes is rapid, efficient, sensitive, specific and easy to operate. It can be used in the rapid field detection of Vibrio parahaemolyticus. Key words: Vibrio parahaemolyticus; Cross-primer amplification technique; Virulence gene