聚合酶链反应方法检测鼠疫耶尔森菌的内部对照研究

目的 避免聚合酶链反应(PCR)方法检测鼠疫菌发生假阴性。方法 通过克隆，将16srRNA引物的扩增产物与鼠疫菌F1抗原基因克隆子相连，并以其作为内部对照模板进行PCR试验。结果 得到了在包含F1抗原基因中连接有16SrRNA扩增产物的质粒，并初步确立了作为内部对照质粒的参照标准浓度。结论 在采用PCR方法检测鼠疫菌时，加入适宜浓度的内部对照质粒作为模板与待检样品同时扩增，可避免假阴性的发生。

Study on the internal control on polymerase chain reaction in Yersinia pestis detection

OBJECTIVE: For the detection of Yersinia pestis by polymerase chain reaction (PCR), internal control (IC) is required in order to prevent false negative results that might be caused by PCR inhibitors. METHODS: F1 antigen was amplified by PCR with primer F1 and the PCR product of primer F1 were cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested with HpaI. The digested plasmid and the PCR products of 16S rRNA were ligated with T(4) DNA ligase before the ligated products were transformed. Isolate plasmid DNA on positive clone and its concentration were measured. Plasmid DNA on different concentration by PCR amplification with primer F1 was analyzed and the standard concentration of IC was determined. RESULTS: Constructing an IC by inserting a 16S rRNA amplicon to the original target DNA between the two primer F1 sites, the size was longer than the target DNA. The standard concentration of IC was determined. CONCLUSION: An optimal IC concentration to increase the reliability of the PCR assays might be used to prevent false negative results and appeared to be useful for detection of Yersinia pestis.