Sensitively and quickly detecting Vibrio vulnificus by real time recombinase polymerase amplification targeted to vvhA gene

Vibrio vulnificus (V. vulnificus) is a Gram-negative bacterium living in warm and salty water. This marine bacterium could produce hemolysin (VVH), which often causes serious gastroenteritis or septicemia when people contact to seawater or seafood containing V. vulnificus. Timely diagnosis is regard as essential to disease surveillance. In this paper, we aimed at developing a quick and sensitive method for the detection of Vibrio vulnificus using real time recombinase polymerase amplification (real time RPA). Specific primers and an exo probe were designed on the basis of the vvhA gene sequence available in GenBank. Target DNA could be amplified and labeled with specific fluorophore within 20 min at 38 ?C. The method exhibited a high specificity, only detecting Vibrio vulnificus and not showing cross-reaction with other bacteria. The sensitivity of this method was 2 pg per reaction (20 ?L) for DNA, or 200 copies per reaction (20 ?L) for standard plasmid. The detection limit (LOD) stated as the target level that would be detected 95% of the time and estimated was 1.58 ? 102 copies by fit of the probit to the results of 8 replicates in different concentration. For quantitative analysis of the real time RPA, the second order polynomial regression was adopted in our study. The results showed the correlation coefficients were raised above 0.98, which suggested this model might be a better choice for the quantitative analysis of real time RPA compared to the routine linear regression model. For artificially contaminated plasma samples, Vibrio vulnificus could be detected within 16 min by real time RPA at concentration as low as 1.2 ? 102 CFU/mL or 2.4 CFU per reaction (20 ?L). Thus, the real time RPA method established in this study shows great potential for detecting Vibrio vulnificus in the research laboratory and disease diagnosis.