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聚合酶链反应快速检出耐甲氧西林金黄色葡萄球菌的研究 |
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莫岚 王其南
【摘要】 目的 建立耐甲氧西林金黄色葡萄球菌的快速检出方法。方法 利用聚合酶链反应(PCR)技术快速检出耐甲氧西林金黄色葡萄球菌(金葡菌)，建立一种从葡萄球菌中快速提取DNA方法，以粗提DNA作为PCR模板，检测编码耐甲氧西林金葡菌青霉素结合蛋白2a(PBP2a)的mecA基因。结果 184株金葡菌用PCR方法及药敏法比较，药敏法鉴定为耐甲氧西林金葡菌(MRSA)58株，仅一株PCR扩增mecA基因阴性。126株甲氧西林敏感的金葡菌(MSSA)中有3株mecA基因阳性。用酶促生物素标记寡核苷酸探针进行Southern blot分析，杂交结果与PCR结果一致，证实533 bp片段为MRSA mecA基因特异性片段。结论 PCR方法能较准确检出MRSA，尤对临界浓度者(MIC为0.5～4 mg/L)较常规方法优越。
【关键词】 聚合酶链反应 葡萄球菌，金黄色 mecA基因
Rapid detection of methicillin-resistant Staphylococcus aureus by using polymerase chain reaction Mo Lan, Wang Qinan. Department of Infectious Diseases, First Affilliated Hospital, Chongqing University of Medical Science, Chongqing 400016
【Abstract】 Objective To identify methicillin-resistant S. aureus (MRSA) with a simple and reliable method, polymerase chain reaction (PCR). Methods A rapid cell lysis procedure was established for the release of DNA from staphylococci. By using crude extract of the strain to be tested as template and 22-mer oligonucleotides as primers, a 533 bp region of mecA gene, We amplified the structural gene of a low affinity penicillin binding protein 2a (PBP2a) by PCR and detected by 2% agarose gel electrrophoresis. Results The results were compared with those obtained by broth double-dilution MIC determination for 184 clinical isolates of S.aureus. 57 of 58 strains of oxacillin-resistant S.aureus were mecA positive as determined by PCR, whereas only one strain of oxacillin-resistant S. aureus was mecA negative. Three strains of 126 oxacillin-susceptible S. aureus were mecA positive. In southern blot ana-lysis of PCR products by using an 18-mer oligonucleotides as a probe, labeled with Bio-11-dUTP, the results of DNA hybridization were correlated with those of PCR test. Conclusion The strains of S. aureus with borderline oxacillin resistance (MIC=0.5～4.0 mg/L) can be accurately identified with PCR method.
【Key words】 Polymerase chain reaction Staphylococcus aureus mecA gene
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