多黏菌素抗性基因mcr-4/5/8实时荧光定量PCR检测方法的建立

A Multiplex Real-time PCR Assay for the Detection of Colistin Resistance Genes mcr-4/5/8

多黏菌素是治疗多重革兰阴性耐药菌最重要的药物之一,近年来,其耐药性逐年持续上升。多黏菌素耐药基因mcr可以通过质粒传播,对人类健康和畜牧业生产造成了严重的威胁。本试验根据GenBank下载的多黏菌素耐药基因mcr-4、mcr-5和mcr-8的基因参考序列,设计特异性引物并验证其特异性,用SYBR Green I染料法建立实时荧光定量检测方法。将mcr-4、mcr-5和mcr-8基因克隆到pMD19-T载体上,构建标准质粒,再以标准质粒为模板建立荧光定量PCR反应的扩增曲线、标准曲线和熔解曲线。结果发现,扩增曲线平行且光滑;标准曲线线性关系良好,扩增效率接近100%,R 2>0.99;标准品熔解曲线呈特异性单峰,表明本次试验构建的方法特异性好。本试验中多黏菌素抗性基因mcr-4、mcr-5和mcr-8实时荧光定量检测方法的建立,可以为未知样品中相应基因的检测提供依据。

Polymyxins are one of the most important drugs in the treatment of multiple gram-negative drug-resistant bacteria,and their drug resistance has been increasing year by year in recent years.Polymyxin-resistant gene mcr can be transmitted through plasmids,which poses serious threat to human health and animal husbandry production.In this study,specific primers were designed and verified according to the gene reference sequences of polymyxin-resistant genes mcr-4,mcr-5 and mcr-8 downloaded from GenBank,and a real-time fluorescence quantitative detection method was established by SYBR Green I dye method.The mcr-4,mcr-5 and mcr-8 genes were cloned into pMD19-T vector to construct standard plasmid,and the amplification curve,standard curve and dissolution curve of fluorescence quantitative PCR reaction were established using standard plasmid as template.The results showed that the amplification curves were parallel and smooth.The standard curve had good linear relationship,and the amplification efficiency was close to 100%,R 2>0.99.The solubility curve of the standard substance showed a specific single peak,indicating that the method constructed in this test had good specificity.The established real-time fluorescence quantitative detection method for polymyxin-resistant genes mcr-4,mcr-5 and mcr-8 in this study can provide a basis for the detection of corresponding genes in unknown samples.