副溶血性弧菌的添加内标的PCR检测方法的建立及评价

Development and evaluation of PCR with internal control in detecting Vibrio parahaemolyticus

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：Developing and evaluating a PCR method that incorporated an internal control（IC） to avoid false negative reactions,for the detection of Vibrio parahaemolyticus.Methods： Genomic comparison analysis was used to analyze the genes toxR and tdh,which were specific for Vibrio parahaemolyticus.The conservative fragment of 900 bp toxR was used as the target sequence.And that of 522 bp tlh was used as the internal control sequence.An IC was designed in such a way that the same primer pair was used to amplify the IC and the target fragment,which were differentiated by the size.PCR parameters were optimized and its reaction system was developed.Results： The specificity of the PCR method with IC constructed was evaluated with V.parahaemolyticus and non-V.parahaemolyticus,the results showed that there were a 900 bp and 560 bp amplicon resulted from V.parahaemolyticus,while there was only a 560 bp IC amplicon from all non-V.parahaemolyticus.The detection limit for DNA was 0.5 pg/μl by PCR with 0.5 fg/μl IC.The PCR method was evaluated with aquatic seafood,and the results demonstrated that it could work effectively.Conclusion： The PCR method with IC could effectively detect V.parahaemolyticus with high accuracy and could especially eliminate false-positive results.