银白色葡萄球菌实时荧光PCR快速检测方法的建立

Development of real-time PCR for the rapid detection of Staphylococcus argenteus

摘要: 本文旨在建立一种银白色葡萄球菌实时荧光PCR检测方法。根据对NCBI数据库中银白色葡萄球菌非核糖体多肽合成酶(NRPS)基因序列的比对分析,设计引物及探针,并基于所设计的引物及探针建立Taq-man 实时荧光PCR的检测方法。结果表明,本实验所建立的方法特异性较强,只能扩增出银白色葡萄球菌,而其他常见菌株的检测结果呈阴性;该方法的灵敏度为 10 pg/uL;在对实验室通过传统方法分离的金黄色葡萄球菌的检测中,发现有两个分离菌株呈阳性,其结果与普通PCR方法完全一致。

Abstract: In this study,a real time polymerase chain reaction assay for detection of newly emerged staphylococcus argenteus was established.Based on the result of multialignment analysis on the published staphylococcus argenteus NRPS sequences on NCBI database,a pair of primers specific and probe were designed,and then a RT-PCR for detection of staphylococcus argenteus was established by utilizing the designed primers and probe. The results showed that detection of common pathogenic bacterias,only staphylococcus argenteus was positive,indicating the established RT-PCR assay was highly specific.The detection limit of 10 pg/uL of the assay was determined. In the laboratory separation of Staphylococcus aureus detection,two positive isolates were found,the results was in complete accord with ordinary PCR method.