荧光定量PCR技术检测副溶血弧菌方法的建立与初步应用

Establishment and Primary Application of Fluorescence Quantitive PCR for Detection of Vibrio parahaemolyticus

摘要: 目的：建立副溶血弧菌SYBR Green I实时荧光定量PCR检测方法。方法：根据副溶血弧菌(VP)的toxR基因序列，应用primer6.0设计了1对特异性引物，进行了最佳引物浓度的确定，建立了标准曲线和溶解曲线，对所建立方法进行了特异性、灵敏度和稳定性测试，并进行了临床样本检测。结果：副溶血弧菌SYBR Green I荧光定量PCR的最佳引物浓度为0.2 µmol/L，标准曲线循环阈值和模板浓度呈良好的线性关系，相关系数R2为0.996，扩增效率E接近100%，溶解曲线特异，最低检出量为2.14个拷贝，特异性和重复性较好。临床检测结果与常规细菌分离鉴定方法符合率100%。结论：建立了特异灵敏的副溶血弧菌实时荧光定量PCR检测方法，对于加强进出口水产品中VP的检验检疫具有十分重要的意义。 Objective: The study developed a SYBR Green Ι real time quantitative PCR for Vibrio parahaemolyticus (VP). Method: According to genome sequences within toxR of VP published in GenBank, a pair of primers was designed by primer6.0. The reaction conditions, sensitivity and specificity of the method were optimized and evaluated. The method was also applied to clinical samples. Result: It was shown that the optimal primer concentration was 0.2 µmol/L. The results also demonstrated that standard curve established was shown a fine linear relationship between threshold cycle and template concentration. The correlation coefficient R2 was 0.996, the efficiency of amplification in E was close to 100%, the dissolution curves were specific and the minimum detectable amount was 2.14 copies. This method has better specificity and reproducibility. The coincidence rate was 100% when compared the results to routine separation and assay method. Conclusion: A SYBR Green 1 fluorescent quantitative PCR for detecting toxR gene of VP was devel- noped which could be very useful for identification and inspection of VP from fishery product in import and export.