耐甲氧西林金黄色葡萄球菌实时荧光PCR检测方法的建立和评价

Research on rapid detection of methicillin resistant staphylococcus aureus by real-time PCR

目的建立实时荧光PCR快速筛查耐甲氧西林葡萄球菌株和耐甲氧西林金黄色葡萄球菌株的方法。方法以耐甲氧西林mecA基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中耐甲氧西林葡萄球菌的实时荧光PCR方法,结合对金黄色葡萄球菌的特异性NUC基因进行实时荧光检测,可鉴定是否为耐甲氧西林金黄色葡萄球菌株。以纸片扩散法作为对照方法,对所建立的实时荧光PCR检测方法检测效果进行初步评价。结果该实时荧光PCR方法的整个检测过程只需要1.5h;对22例临床样本进行检测,所建立的荧光PCR方法比纸片法多检出2例耐甲氧西林菌株;对耐甲氧西林金黄色葡萄球菌的检测结果一致。结论本研究建立的实时荧光PCR检测耐甲氧西林金黄色葡萄球菌的方法不仅能实现耐甲氧西林金黄色葡萄球菌的快速检测,更可能为指导临床用药提供有价值的参考。

Objective To establish a real-time polymerase chain reaction (real-time PCR) assay for rapid detection of methicillin resistant staphylococcus aureus(MRSA). Methods The special sequence of mecA gene of methicillin resistant staphylococcus was amplified and characterized with a pair primers and TaqMan probe. With detecting the nuc gene of staphylococcus aureus, the method of detecting MRSA was developed. Comparing with KB-Test, the method was evaluated by detecting 22 samples. Results The real-time PCR method was designed to sensitively detect and identify MRCoNS and MRSA. The total assay could be completed in 1.5 hours. 6 samples were positive detected by the real-time PCR method, two more than KB-Test. And the result for MRSA strain was coincident by the two methods. Conclusion RT-PCR detection methods provide a special, sensitive, rapid, reproducible and accurate method for quantitative detection of MRSA, and provide an important reference for clinical medication.