期刊文献+

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
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摘要 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.
作者 尚伟 代稳 陈北方 王玫 邹大阳 仓宝成 Shang Wei, Dai Wen, Chen Beifang, Wang Mei, Zou Dayang, Cang Baocheng( Laboratory Center of Chinese PLA 153 Hospital, Zhengzhou 450042, China ( Shang Wei, Dai Wen, Chen Beifang, Cang Baocheng); Zhengzhou University of Light Industry, Zhengzhou 450042, China (Wang Mei) ; PLA Institute for Disease Control and Prevention, Beijing 100071, China ( Zou Dayang))
出处 《中华微生物学和免疫学杂志》 CSCD 北大核心 2018年第9期710-715,共6页 Chinese Journal of Microbiology and Immunology
基金 国家自然科学基金(61704157)
关键词 黏菌素耐药 mcr-1基因 PCR检测 TAQMAN探针 Colistin resistance mcr-1 gene PCR TaqMan probe
作者简介 通信作者:邹大阳,Email:zoudayang@yeah.net,电话:010-66948751;;仓宝成,Email:cbcl53jyk@163.com.电话:0371-60653530
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