应用PCR方法直接检测痰中耐甲氧西林葡萄球菌

目的建立直接用痰标本通过PCR方法快速检测耐甲氧西林葡萄球菌的方法。方法利用聚合酶链反应技术（PCR）快速检出耐甲氧西林葡萄球菌，建立一种从痰中快速提取DNA方法，以粗提DNA作为PCR模板，检测编码耐甲氧西林葡萄球菌青霉素结合蛋白2a（PBP2a）的mecA基因和细菌中均有的16SrRNA基因。结果364份临床痰标本经PCR方法检出mecA基因的38份，药敏法检出耐甲氧西林的葡萄球菌33份；38份mecA基因阳性的痰标本中有31份药敏法检测为耐甲氧西林的葡萄球菌，药敏法检出耐甲氧西林的葡萄球菌33份痰标本中有2份mecA基因阴性。16SrRNA基因片段在PCR中作为内部对照避免了假阴性结果的出现。结论用PCR直接检测痰中的mecA基因，是判断痰中是否含有耐甲氧西林的葡萄球菌的一种快速有效的方法。

Directly identification of methicillin-resistant staphylococci in sputum by using polymerase chain reaction

Objective To identify methicillin- resistant staphylococci in sputum specimens with polymerase chain reaction (PCR) using sputum directly. Methods A rapid procedure for the release of DNA from staphylococci in sputum was established. By using crude extract of the strain in sputum as template ,we amplified the structural gene(393bp region of mecA gene) encoding a low affinity penicillin binding protein 2a(PBP2a) and another gene(876bp region of 16S rRNA) commonly distributing in all strains with PCR method and 2% agarose gel electrophoresis. Results Thirty- eight mecA- positive strains were detected from 364 clinical samples by PCR, whereas 33 methicillin- resistant Staphylococcus were determined by drug sensitive test; 31 of 38 mecA- positive staphylococci were methicillin- resistant, 2 of 31 methicillin- resistant staphylococci were mecA negative. The 876- bp region of the 16S rRNA gene was used as a PCR internal control. Conclusion It is a rapid and effective method to identify methicillin- resistant staphylococci by detecting mecA gene in sputum directly.