应用DNA探针检测甲氧西林耐药葡萄球菌

Rapid detection of methicillin-resistant staphylococci by DNA probe

目的建立一种快速检测葡萄球菌和甲氧西林耐药葡萄球菌的斑点杂交技术。方法设计金黄色葡萄球菌nuc基因、甲氧西林耐药mecA基因、葡萄球菌tuf基因的特异引物,用聚合酶链反应合成其特异DNA探针,并用生物素标记,分别与固定在硝酸纤维素膜上的标准菌株和临床分离株模板DNA杂交,观察其敏感性和特异性。结果3对引物分别扩增出270bp、310bp、370bp3种DNA探针,均具有高度特异性。50株金黄色葡萄球菌tuf、nuc基因均为阳性,mecA基因阳性者22株。30株表皮葡萄球菌tuf基因均为阳性,nuc基因均为阴性,mecA基因阳性者9株。而其他非葡萄球菌与3种DNA探针杂交结果均为阴性。该方法可检测出1ng细菌DNA。结论斑点杂交技术检测耐甲氧西林葡萄球菌快速、有效,具有较高的应用价值。

Objective To establish a dot blot hybridization technique for rapid detection of staphylococci and methicillin-resistant staphylococci. Methods Three pairs of primers were designed according to nuc gene of staphylococcus aureus, mecA gene of methicillin-resistance, tuf gene of staphylococci. Specific DNA probes were synthesized by polymerase chain reaction and labeled with biotin. The bacterial DNA inoculated on nitrocellulose filter was hybridized with these probes. The sensitivity and specificity were detected. Results The DNA probes with 270bp,310bp and 370bp were amplified by the three pairs of primers respectively. The probes were specific. Among 50 clinical isolates of staphylococcus aureus tuf and nuc gene were all positive and mecA gene in 22 isolates were positive. Positive rate of tuf, nuc and mecA gene in 30 staphylococcus epidermidis were 100% ,0 and 30% （9/30） respectively. No hybridization in other nonstaphylococci occurred. The established method could detect as low as 1 ng of bacterial DNA. Conclusion The dot blot hybridization is of high value in rapid, effective identification of methicillin-resistant staphylococci.