caf1基因为靶标的鼠疫检测PCR法的改进

摘要

Objective To identify the causes of nonspecific bands in the detection of a industry standard caf1 gene by polymerase chain reaction(PCR),and to propose a solution to this problem. Methods A total of 112 strains were selected for the experiment, including 40 strains of Yersinia pestis, 72 strains of non-Yersinia pestis;DNA was extracted,and caf1 gene was amplified by PCR;seven non-specific strips were recovered,purified and TA cloning and sequencing; the primer of the caf1 gene was redesigned and validated using all of the strains. Results Using the industry standard caf1 gene primer,DNAs of 40 Yersinia pestis and 72 non-Yersinia pestis were amplified by PCR, 58 non-Yersinia pestis could be amplified with non-specific bands, they were about 400, 500, 600, 700, 800, 900, 1 000 bp. By TA cloning and sequencing, the non-specific bands in the downstream of the industry standard caf1 primer and its reverse complement were amplified. Using the new designed caf1 primer to amplify, 72 non-Yersinia pestis strains showed no non-specific bands. Conclusion Non-specific bands has been amplified in the screening of Yersinia pestis using the primer of the industry standard caf1, and the new caf1 primer can effectively avoid this problem and improve the accuracy of detection.%目的 查明行业标准推荐caf1基因引物(简称行标caf1引物)进行PCR检测时,在一些非鼠疫菌上出现非特异性条带的原因,并提出解决办法.方法 共选取112株菌进行试验,其中鼠疫菌40株、非鼠疫菌72株;提取菌株DNA,并对caf1基因进行PCR扩增;选择出现非特异性条带的7株菌株,进行切胶回收、纯化以及TA克隆测序;针对caf1基因重新设计引物,对被试菌株进行验证.结果 使用行标caf1引物扩增40株鼠疫菌,均未出现非特异性条带,扩增72株非鼠疫菌,有58株扩增出400、500、600、700、800、900、1000 bp的非特异性条带.经克隆和测序,在非特异性产物的基因序列中均出现行标caf1引物的反向引物序列及反向引物的反向互补序列.重新设计caf1引物,扩增72株非鼠疫菌,均未出现非特异性条带.结论 行标caf1引物在筛查鼠疫菌时会出现非特异性条带,使用新caf1引物可有效避免这一情况,提高检测的准确性.