等温扩增检测耐药基因MECA方法的建立及其临床应用

Establishing a method of loop-mediated isothermal amplification for detecting resistant gene mecA rapidly in clinic sample

目的建立环介导的等温扩增快速检测葡萄球菌耐药基因MECA的方法。方法利用包被有SPA单克隆抗体的磁珠富集样品中的金黄色葡萄球菌,快速提取其DNA,用环介导的等温扩增反应(LAMP)扩增金黄色葡萄球菌耐药基因MECA,扩增条件为65°C保温1H。在反应过程中,荧光探针与MECA基因的扩增产物互补序列结合,产生荧光,通过检测反应管中的荧光值来判定结果。结果该方法的最低检测限为10CFU/ML,与M-H培养基苯唑西林药敏试验结果相比较,灵敏性99%,特异性90·9%;而与PCR方法比较,灵敏性100%,特异性100%;试验时间缩短为1H。结论该方法具有灵敏度高,特异性强,操作简便快速,适用于检测临床标本中的MRSA。

Objective To establish a method of Loop-mediated isothermal amplification （LAMP） with molecular beacon for detection of mecA gene from MRSA in clinic samples. Methods After a rapid DNA extraction by immunomagnetic enrichment in S. aureus through anti-spa antibody-coated paramagnetic beads, mecA gene of MRSA was amplified by LAMP. At first,all reagents were mixed in a single tube, the tube was then held at 65℃ for 45 min. The resulting amplicons were detected by measuring fluorescence signal of hybridization of molecular beacon to amplicons in the reaction tube. Results The assay had a minimum detection of 10 CFU/ml Compared with MH agar containing oxacillin, its sensitivity and specificity were 99% and 90. 9% respectively; compared with PCR, its sensitivity and specificity were both 100%. Conclusions This LAMP-based assay is simple, rapid, sensitive and specific; a result is available in 1 h for a clinic swab specimen that contains a corresponding amount of DNA