多重分子信标环介导等温扩增快速检测耐甲氧西林金黄色葡萄球菌

Multiplex Loop-mediated Isothermal Amplification (LAMP) with Molecular Beacon for Rapidly Detecting Meticillin-resistant Staphylococcus aureus

目的建立多重分子信标环介导的等温扩增快速检测葡萄球菌耐药基因mecA和种特异性基因femA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,用多重环介导等温扩增反应(LAMP)同时扩增金黄色葡萄球菌耐药基因mecA和femA,扩增条件为65℃保温1 h;在反应过程中,两种分子信标荧光探针分别与mecA和femA基因的扩增产物互补序列结合,探针上的FAM和TET荧光分子产生两种荧光,最后检测反应管中的两种荧光值。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,灵敏度99.0%,特异度90.9%。结论该方法具有灵敏度高、特异性强、操作简便快速,适用于直接快速基因检测临床样品中MRSA。

OBJECTIVE To establish a method of multiplex loop-mediated isothermal amplification （LAMP） with molecular beacon for detecting mecA gene and femA gene of MRSA in clinic sample. METHODS After a rapid samples conditioning of immunomagnetic enrichment in S. aureus by anti-SPA antibody-coated paramagnetic beads, mecA gene and femA gene of MRSA in DNA extracts were amplified using LAMP, the whole procedure was quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture was held at 65℃. The resulting amplicons were detected by measuring fluorescence signal of hybridization of molecular beacon to amplicons of the reaction tube. RESULTS The assay had a detection limit of 10 CFU/ml MRSA with a 60 min incubation time at 65℃ ; it＇s specificity was validated on 4 Staphylococcus species and 3 nonstaphylococcus species; compared to culture-based techniques, its sensitivity and specificity were 99.0% and 90. 9%, respectively. CONCLUSIONS This LAMP-based assay is simple, rapid, sensitive and specific; a result is available in 60 min for a clinic swab specimen that contains a corresponding amount of DNA available for testing.