高分辨率熔解曲线分析法检测食源性副溶血性弧菌

rapid real-time PCR method for detecting Vibrio parahaemolyticus by high resolution melting curve analysis(HRM) was developed. A pair of specific primers was used targeting the toxR gene of Vibrio parahaemolyticus (VP).After optimizing the conditions, the specificity of the method was validated with 10 target strains and 10 non-target strains,the solutions with different copies of purified amplification products were analyzed by the method to evaluation its sensitivity,the reproducibility of the method was also evaluated.The method was also applied to the deteciton of 90 seafood samples.The results showed that :(1)the developed HRM real-time PCR assay protocol showed a good specificity by detecting only VP with the Tm value of 76.64 ℃±0.57 and was not affected by other nomal seafood pathogens such as Vibrio vulnificus, Vibrio cholerae,Staphylococcus aureus,Salmonela, Shigella flexneri et al.（2）the sensitivity of detection of the constructed plasmids were 3.50×102 copies /ml.(3) the developed protocol of HRM real-time PCR assay showed a high reproducibility, the average Tm values were 76.53 ℃±0.35 and 76.74 ℃± 0.52,the sample's variations (CVs) were 0.56±0.42 ％ and 1.11±0.73 ％ whin the sample and between tests ,respectively.(4) the detection of 90 live seafood samples displayed a better detection rate of 18.89 % than a 14.44 % of the traditional culture method.The established real-time PCR assay in this study has sufficient specificity,high ensitivity and good reproducibility,and can also quantitatively detect VP in only 3 hours.It is rapid,simple and cheap for detecting VP and would be useful in food microbiology laboratory.