等位基因特异性多重聚合酶链反应方法用于快速检测结核分枝杆菌利福平耐药株

目的建立检测耐利福平(RFP)结核分枝杆菌的等位基因特异性多重聚合酶链反应(multiplex allele specific polymerase chain reaction,MAS-PCR)方法,快速、特异地检测rpoB基因核心突变区的主要突变,用于快速诊断结核分枝杆菌对利福平的耐药性。方法根据结核分枝杆菌的rpoB序列,分别设计出3对特异性寡聚核苷酸引物,采用MAS-PCR技术,分别检测rpoB基因上531、526、516这3个最常见的突变位点的突变。结果对临床分离的利福平敏感株(15株)及利福平突变株(81株)进行检测,以直接测序结果为参照,对利福平耐药株的总检出率为81.5%(66/81)。结论MAS-PCR方法敏感、特异,可快速、简便地检测结核分枝杆菌rpoB基因突变,有利于耐药结核分枝杆菌的快速检测。

A new multiplex allele-specific polymerase chain reaction assay for detection of rifampin-resistant Mycobacterium tuberculosis

Objective To develop a new multiplex allele-specific polymerase chain reaction(MAS-PCR) assay to detect the main mutations in the rifampin resistance dependent region, which has been reported to account for the majority of clinic Mycobacterium tuberculosis resistant to rifampin. Methods Based on the sequence of rpoB gene, three specific primers were designed for the MAS-PCR to detect the most common mutations in codons 531,526,516 of rpoB gene. Results The purified DNA preparations of 91 clinical strains of Mycobacterium tuberculosis were used to optimize the PCR. The mutations in codon 531,526,516 were detected by the MAS-PCR.Compared with the results of direct sequencing of rpoB gene, no mutation was detected in the sensitive strains. For rifampin-resistant strains, the total sensitivity was 81.5%(66/81). Conclusions MAS-PCR is a new molecular method with a high sensitivity and specificity, which can be used to detect the 3 main mutations of rpoB gene rapidly and economically. It can be used in clinical laboratories to detect the rifampin-resistant strains of Mycobacterium tuberculosis.