二重实时PCR快速检测耐甲氧西林金黄色葡萄球菌  

Rapid detection of methicillin-resistant Staphylococcus aureus with duplex real-time PCR assay

作　　者：王艳[1] 叶冬青[1] 李家斌[2] 张胜权[3] 

机构地区：[1]安徽医科大学公共卫生学院,合肥230032 [2]安徽医科大学第一附属医院,合肥230022 [3]安徽医科大学分子生物学教研室,合肥230032

出　　处：《中国抗生素杂志》2007年第4期225-228,255,共5页Chinese Journal of Antibiotics

摘　　要：目的建立二重实时聚合酶链反应(duplex real-time PCR)快速检测耐甲氧西林金黄色葡萄球菌(MRSA)。方法采用二重SYBR Green实时PCR快速检测MRSA的决定基因mecA和金葡菌的种特异性基因nuc,经熔解曲线分析鉴定产物。结果所有MRSA菌株的熔解曲线均呈现mecA、nuc基因特异性的峰,甲氧西林敏感的金葡菌仅有nuc峰,耐甲氧西林的表葡菌仅有mecA峰,甲氧西林敏感的表葡菌与其他菌种的菌株无特异峰出现;当MRSA菌浓度达102cfu/ml时就可检出。在131株金葡菌中,30.53%(40/131)耐药;二重实时PCR扩增mecA基因29.01%(38/131)阳性,nuc基因100%阳性。单引物与二重实时PCR两者阳性扩增符合率为100%。结论对mecA、nuc基因进行二重实时PCR能准确、快速地鉴定耐甲氧西林的金葡菌和凝固酶阴性的葡萄球菌。Objective To establish a duplex real-time polymerase chain reaction （PCR） assay for rapid detection of methicillin-resistant Staphylococcus aureus （MRSA）. Method A duplex SYBR Green realtime PCR assay targeting the rnecA gene and a Staphylococcus aureus specific marker-nuc gene and identifying PCR products through melting curve analysis was used to rapidly identify MRSA. Results All MRSA strains tested in the study presented mecA and nuc gene specific peaks in the melting curve analysis ; methicillinsusceptible Staphylococcus aureus （MSSA） strains only had a nuc peak, methicillin-resistant Staphylococcus epiderrnidis （MRSE） strains only had a rnecA peak, and methicillin-susceptible Staphylococcus epidermidis （MSSE） strains and strains of species other than staphylococci had no peak. MRSA strains could be detected, by use of this duplex real-time PCR in an 10^2cfu/ml inoculum. Positive rate of drug sensitivity test was 30. 53% （40/131） among 131 S. aureus isolates tested, of which 29.01% （38/131） were positive for mecA gene and all were positive for nuc gene in the duplex real-time PCR. The agreement between simplex and duplex real-time PCR was 100% for positive results. Conclusion This study shows that the duplex real-time PCR assay with the primers for to mecA and nuc genes is a valuable tool for rapid and precise identification of MRSA and methicillin-resistant coagulase-negative staphylococci （MRCNS）.

关 键 词：实时聚合酶链反应 耐甲氧西林金葡菌 MECA基因 nuc基因 

分 类 号：R378.11[医药卫生—病原生物学]