Sensitive and Rapid Detection of the Plasmid-Encoded Colistin-Resistance Gene mcr-1 in Enterobacteriaceae Isolates by Loop-Mediated Isothermal Amplification.
Sensitive and Rapid Detection of the Plasmid-Encoded Colistin-Resistance Gene mcr-1 in Enterobacteriaceae Isolates by Loop-Mediated Isothermal Amplification.
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通过环介导等温扩增灵敏快速检测肠杆菌科分离株中质粒编码的粘菌素抗性基因 mcr-1。
DOI：
10.3389/fmicb.2017.02356
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发表时间：
2017
影响因子：
5.2
通讯作者：
Huang L
中科院分区：
文献类型：
--
作者：
Zou D;Huang S;Lei H;Yang Z;Su Y;He X;Zhao Q;Wang Y;Liu W;Huang L
肠杆菌科中质粒编码的粘菌素耐药基因mcr-1的出现代表了临床环境中感染治疗的新威胁。需要一种灵敏、快速的分子检测方法来检测临床分离株中的mcr-1基因，以控制该基因的传播。在这项研究中，我们建立了一个环介导等温扩增（LAMP）检测方法，用于快速检测mcr-1基因。将该测定法应用于培养的细菌和加标的人粪便。实时监测浊度和显色可视化用于评估反应结果。测定了LAMP反应中用于检测mcr-1基因的引物的特异性和灵敏度。所有20株不含mcr-1基因的临床耐药分离株检测结果均为阴性，表明LAMP引物具有高度特异性。LAMP的灵敏度为0.2pg/μL DNA，是PCR的10倍。当应用于加标mcr-1阳性大肠埃希菌的人粪便时，该试验也具有决定性。在中国北京一家大型医院的临床筛选期间，从556株肠杆菌科分离株中鉴定出7株阳性分离株。总之，我们开发的LAMP检测方法可用于临床环境中的mcr-1基因检测。
The emergence of the plasmid-encoded colistin-resistance gene mcr-1 in Enterobacteriaceae represents a new threat to the treatment of infection in the clinical setting. A sensitive and rapid molecular method for detection of the mcr-1 gene in clinical isolates is needed to control the spread of this gene. In this study, we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the mcr-1 gene. This assay was applied to cultured bacteria and spiked human stools. Real-time monitoring of turbidity and chromogenic visualization were used to assess the reaction results. The specificity and sensitivity of the primers in the LAMP reactions for detection of the mcr-1 gene were determined. All 20 clinically resistant isolates without the mcr-1 gene tested negative, indicating the high specificity of the LAMP primers. The sensitivity of LAMP, with a detection limit of 0.2 pg/μL DNA, was 10-fold greater than that of polymerase chain reaction (PCR). The assay was also conclusive when applied to human stools spiked with mcr-1-positive Escherichia coli. During clinical screening in a major hospital in Beijing, China, seven isolates were identified as positive from the 556 Enterobacteriaceae isolates. In conclusion, the LAMP assay we developed was useful for detection of the mcr-1 gene in the clinical setting.