聚合酶链反应检测耐甲氧西林金黄色葡萄球菌mecA基因

Detection of Methicillin-resistant Staphylococcus Aureus mecA gene by polymerase chain reaction

建立聚合酶链反应（ｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ，ＰＣＲ）检测耐甲氧西林金黄色葡萄球菌（ｍｅｔｈｉｃｉｌｌｉｎ－ｒｅｓｉｓｔａｎｔＳ．ａｕｒｅｕｓ，ＭＲＳＡ）ｍｅｃＡ基因的技术。四种ＤＮＡ提取方法检测灵敏度依次为超声裂解法（５×１０５ＣＦＵ／ｍｌ）、溶菌酶表面活性剂法（５×１０６ＣＦＵ／ｍｌ），表面活性剂法（１×１０７ＣＦＵ／ｍｌ）和直接煮沸法（１×１０８ＣＦＵ／ｍｌ），直接煮沸法具有简便性。引物的正链位于１８１～２００、负链位于３１１～３３０，序列分别为５＇－ＧＡＡＡＴＧＡＣＴＧＡＡＣＧＴＣＣＧＡＴ，５＇－ＧＣＧＡＴＣＡＡＴＧＴＴＡＣＣＧＴＡＧＴ，其扩增产物长度为１５０ｂｐ。五种常见菌证明本法具有较高特异性。苯唑青霉素ＭＩＣ水平与ｍｅｃＡ基因之间具有很好的相关性，ＭＩＣ≥４μｇ／ｍｌ的２２株菌均检出ｍｅｃＡ基因，提示苯唑青霉素常规检测ＭＲＳＡ的可行性。ＰＣＲ方法对于隐匿型耐药株的检出具有重要价值。

A polymerase chain reaction (PCR) assay test was developed for the detection of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA). The sensitivities in four of DNA extraction methods from Staphylococcus aureus were sequentially lysis cell by ultrasonics (5×105 CFU/ml), lysozymesurfactant (5×106 CFU/ml), surfactant (1×107 CFU/ml) and directly boiling (1×108 CFU/ml). There was a simplicity in the DNA extraction by directly boiling. A pair of primers that corresponded to nucleotides 181 to 200 as the sense strand with sequence 5'-GAAATGACTGAACGTCCGAT and 311 to 330 as the antisense strand with 5'-GCGATCAATGTTACCGTAGT were selected for amplifing a 150 bp-long segment of the mecA gene. The better specificity was shown by the results of 5 species strains tested. There was an excellant relationship between the presence of the mecA gene and the level of bacteria to oxacillin MICs. The fact that the mecA gene were detected from all of 22 strains with the MIC≥4μg/ml to oxacillin suggested the reliability for routinely determing MRSA by the MIC levels to oxacillin. There was most importance in detecting cryptically methicillin-resistant strains by PCR technique.