黏菌素耐药细菌mcr-1基因TaqManPCR快速检测方法的建立

Real-time quantitative PCR assay with TaqMan probe for rapid detection of colistin resistance mcr-1 gene

目的 建立一种快速灵敏准确的分子检测方法用来检测临床分离菌株中的mcr-1基因.方法 根据mcr-1基因的序列设计一对特异性引物和一条TaqMan探针,同时构建含有mcr-1基因片段的重组质粒作为阳性标准品,采用TaqMan探针荧光定量PCR方法检测耐药基因mcr-1,并对方法的敏感性、重复性和特异性进行评价.结果 TaqMan探针荧光定量PCR结果显示起始模板量与Ct值之间存在良好的线性关系（R2〉0.999）,检出下限为10拷贝/μl,比常规PCR敏感性高100倍;特异性检测显示只有含mcr-1基因的菌株结果为阳性,其余菌株为阴性;组内及组间重复性试验的变异系数均小于1％.利用所建立的方法对150株临床分离的耐药菌株进行检测,检测到2株菌株携带mcr-1耐药基因,2株菌株鉴定均为大肠杆菌.结论 建立的TaqMan探针荧光定量PCR检测耐药基因mcr-1的方法具有特异性强、敏感性高、重复性好的优点,可用于临床检验上特异性检测携带mcr-1基因的临床耐药菌株,为临床药物治疗提供更好的依据.

Objective To establish a sensitive real-time quantitative PCR assay with TaqMan probe for rapid detection of mcr-1 gene in clinical isolated strains. Methods According to the mcr-1 gene sequence, a pair of specific primers and a TaqMan probe were designed. Moreover, a recombinant plasmid with mcr-1 gene was constructed as the positive standard. TaqMan probe-based fluorescence quantitative PCR assay was used to detect the colistin resistance gene mcr-1. The sensitivity, repeatability and specificity of the assay were evaluated. Results There was a good linear relationship between the initial template amount and Ct value （R2〉0. 999）. The lower limit of detection was 10 copies/μL, which was 100 times more sensitive than the conventional PCR. Results of test for specificity showed that only the strains carrying the mcr-1 gene were positive, while the remaining strains were negative. Coefficients of variation of intra-and inter-group repeatability tests were less than 1%. Two out of 150 clinical isolated strains carried mcr-1 re-sistance gene and both of them were identified as Escherichia coli. Conclusion TaqMan probe-based fluo-rescence quantitative PCR for the detection of colistin resistance gene mcr-1 was established with strong spe-cificity, high sensitivity and good repeatability. It could be used for the specific detection of clinical drug-re-sistant strains positive for mcr-1 gene and provide reference for pharmacotherapy.