摘要
目的 建立和应用泰泽菌巢式PCR检测方法。方法 参照日本学者Goto检测泰泽菌 16SrDNA序列设计的两对引物 ,对PCR的检测条件进行了改进和优化。结果 巢式PCR反应的外引物能引导扩增出一条 6 2 5bp的DNA片段 ,内引物则能引导扩增出一条 196bp的DNA片段。此 6 2 5bp和 196bpDNA片段的大小均与Goto报道的结果完全一致。试验证明 196bp的阳性带为泰泽菌 16SrDNA特异性保守片段。通过将泰泽菌阳性肝组织标本经系列稀释的试验表明 ,该PCR方法的检测灵敏度为 2个菌。另外 ,将阳性对照标本扩增的 6 2 5bpDNA片段作核酸序列测定 ,发现其碱基序列与Goto报道的泰泽菌MSK株及RJ株 16SrDNA序列均有 99%同源性。结论 根据上述结果 ,我们应用本方法对普通级小鼠、大鼠、金黄色地鼠肝组织标本各 5份、普通级豚鼠肝组织标本 10份、沙鼠肝组织标本 10份、MHV3 感染的小鼠肝组织 1份进行了初步检测 。
Objective To establish a nest PCR method and its application for testing 16S rDNA of Clostridium Piliforme. Method The nest PCR condition was modified on the basis of Goto's report. Results The outside pair of primers can lead to the synthesis of a 625*!bp DNA fragment while the inside pair of primers can lead to a 196 bp DNA fragment. Both amplified fragments are exactly the same size as Goto 's report. The 196bp DNA fragment has been demonstrated to be the specific sequence conserved in Clostridium Piliforme in our experiment. Our PCR assay is of great sensitivity, detecting even 2 organisms in positive sample. The sequence of 625 bp DNA fragment amplified in our positive control is 99% homologous to both the MSK and RJ strains as reported by Goto. Conclusion The nest PCR assay established in our laboratory can be used to amplify specifically 16S rDNA of Clostridium Piliforme. 5 liver samples each from mouse, rat, golden hamster, 10 liver samples each from gerbile, guinea pig bred in conventional environment and 1 liver sample infected with MHV3 from mouse bred in clean environment have been tested for Clostridium Piliforme by this method. No latently infected animals have been identified among the 36 samples.
出处
《中国实验动物学杂志》
2001年第4期224-227,共4页
Chinese Journal of Laboratory Animal Science
