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目的:建立并评价检测副溶血性弧菌(Vibrio parahaemolyticus)的添加内标(internal control,IC)的PCR方法。方法:利用生物信息学方法,分析副溶血性弧菌中特异基因toxR和tlh,设计引物,toxR中900 bp的特异序列为目的片段;tlh中522bp的特异序列为添加片段,利用复合引物法构建IC,建立PCR检测体系。结果:本研究成功构建添加IC的PCR检测体系,利用该体系对副溶血性弧菌和非副溶血性弧菌进行检查,结果显示,以副溶血性弧菌为模板的PCR反应可扩增到900 bp和560 bp的特异片段,而以非副溶血性弧菌为模板的反应扩增到一条560 bp的IC片段。该方法对DNA灵敏度的检测极限是0.5 pg/μl,此时IC的最佳添加浓度是0.5 fg/μl。并对多份水产品的检测结果也证实其能起到很好的指示假阴性作用。结论:添加内标的PCR方法能够准确检测水产品中副溶血性弧菌,排除假阴性干扰。
OBJECTIVE: To establish and evaluate a PCR method for the detection of Vibrio parahaemolyticus added with internal control (IC). Methods: Using bioinformatics methods to analyze the specific genes toxR and tlh in Vibrio parahaemolyticus. The specific primers of 900 bp were designed in toxR. The specific sequence of 522 bp in tlh was added fragment, IC, the establishment of PCR detection system. Results: In this study, a PCR detection system with IC was successfully constructed. Vibrio parahaemolyticus and non-parahaemolyticus were examined by this system. The results showed that the PCR reaction with Vibrio parahaemolyticus as a template could be amplified to 900 bp and 560 bp of specific fragments, while non-parahaemolyticus as a template reaction amplified a 560 bp IC fragment. The limit of detection of DNA sensitivity by this method is 0.5 pg / μl, and the optimal concentration of IC for this method is 0.5 fg / μl. And the test results of many aquatic products also confirmed that it can play a very good indicator of false negative effect. Conclusion: The internal standard PCR method can accurately detect Vibrio parahaemolyticus in aquatic products and eliminate false negative interference.