SYBR GreenⅠ实时荧光定量多重PCR快速筛查葡萄球菌中常见大环内酯类耐药基因

Multiplex Real-time SYBR GreenⅠPCR Assay for Rapid Screen Common Macrolides Resistance Genes in Staphylococci

目的评价SYBRGreenI实时荧光定量多重PCR结合熔解曲线分析快速筛查葡萄球菌常见大环内酯类耐药基因的可行性,利用此筛查方法了解深圳地区葡萄球菌中大环内酯类耐药基因的分布情况。方法选取经测序鉴定携带有大环内酯类耐药基因ermA,ermB,ermC,msrA的葡萄球菌,用一个包括这四种耐药基因引物的SYBRGreenI多重实时荧光定量PCR检测,经荧光定量曲线和熔解温度曲线(Tm值)确证和反应条件优化后建立针对大环内酯类常见耐药基因的初筛体系;随机选取临床136株葡萄球菌,利用此初筛体系对136株葡萄球菌是否携带常见大环内酯类耐药基因进行检测,并对检测结果和表型、电泳、测序结果进行比较,从而对此方法进行评价。对携带有大环内酯类耐药基因的细菌进行基因型别的鉴定,以了解大环内酯类耐药基因的分布情况。结果此SYBRGreenI实时荧光定量多重PCR初筛体系检测葡萄球菌常见大环内酯类耐药基因与表型检测比较达到94.8(129/136)的一致性,与琼脂糖凝胶电泳结果和测序结果比较达到100的一致性。136株葡萄球菌检出msrA,ermA,ermB,ermC四种耐药基因,耐药基因总检出率为75.7(103/136),并发现多株携带多种大环内酯类耐药基因的葡萄球菌。结论SYBRGreenI实时荧光定量多重PCR可以快速准确地筛查葡萄球菌是否携带有常见的大环内酯类耐药基因;深圳地区葡萄球菌大环内酯类耐药率较高,主要耐药基因是ermC56.6(77/136),msrA24.2(33/136),ermA12.5(17/136)。

Objective To assess Multiplex real-time SYBR Green I PCR combining melting temperature assay for rapid screen common macrolides resistance genes in staphylmcocci and investigate the distribution of macrolides resistance genes of staphylococci in that district. Methods Applied a Multiplex real time SYBR Green I PCR system including ermA,ermB, ermC and msrA primer pairs to detect macrolides resistence genes in staphylococci harboring these resistence genes confirmed by sequencing and optimize that PCR system by fluorescent quantitation and melt temperature（Tm） curve assay. Used that optimized screen system to detect 136 staphylococci and compared with electrophoresis,sequencing and pheno- type detected result to evaluate that method. Meanwhile, confirming genotype in resistant staphylococci. Results That multiplex real-time SYBR Green I PCR screen system reached 94. 8%（129/136） accord with phenotype detection and 100% accord with agarose electrophoresis and sequencing result, ermA,ermB, ermC and msrA were detected in 136 staphylococciand. Positive result occupys 75.7% （103/136）. Conclusion Multiplex real-time SYBR Green I PCR combining melting temperature assay can screen rapidly common macrolides resistence genes in staphylococci. The resistance incidence is high in that district and the predominant resistance genes are ermC 56.6%（77/136）,msrA 24.2%（33/136） and ermA 12.5%（17/136）.