PCR方法检测猕猴奇异变形杆菌

摘要

目的 利用PCR检测猕猴奇异变形杆菌,为该病菌的鉴定、临床诊断、治疗及流行病学调查等提供参考.方法 从猕猴粪便中分离奇异变形杆菌并进行鉴定,纯培养后提取基因组DNA,经PCR扩增、凝胶电泳检测、基因序列测序后,序列提交BLAST比对分析.用该菌株检验PCR方法的特异性和敏感度,利用PCR方法和传统分离培养法同时对80只猕猴进行检测.结果 在分离得到的猕猴奇异变形杆菌中扩增出225 bp的基因序列(GenBank登录号为JQ040548,其与GenBank中奇异变形杆菌AM942759的同源性为98.2%),而其它5株非奇异变形杆菌的PCR扩增结果为阴性,表现出极好的特异性.纯化培养的奇异变形杆菌利用PCR方法检测灵敏性,其敏感度达80 cfu/mL.在80只猕猴中,共检测出了2份阳性样品,阳性率为2.5%,检测结果与传统培养方法一致.结论 该PCR方法能迅速、准确地检测猕猴奇异变形杆菌,在临床具有良好的实用性.%In order to provide data for Proteus mirabilis identification , clinical diagnosis , treatment and epidemiological investigation in Macaca mulatto, the Proteus mirabilis from Macaca mulatto was detected by PCR method in this study . Proteus mirabilis was isolated and identified from feces of Macaca mulatta, and the DNA was extracted from cultured strains and then amplified by PCR . The products were analyzed by gel electrophoresis and then sequencing . The sequence of Proteus mirabilis was identified by BLAST . The specificity and sensitivity of PCR were assessed . Eighty samples of Macaca mulatta were screened for Proteus mirabilis by PCR method and traditional method of isolation and identification simultaneously . Results showed that the sequence (225 bp) of Proteus mirabilis was amplified from Macaca mulatta and submitted to GenBank (No . JQ040548); homology analysis showed that the strain shared 98 .2% similarity with the Proteus mirabilis (AM942759). The specificity was identified by negative amplification of other 5 non-Proteus mirabilis strains. The sensitivity of PCR was 80 cfu/mL . The PCR method was comparable with traditional method of isolation and identification as the positive rate of Proteus mirabilis in Macacamulatta detected by that was 2 .5% (2/80) . The PCR method was useful for its rapid and accurate detection of Proteus mirabilis in Macacamulatta, which would be applicable in clinic.