一次性完成结核分枝杆菌鉴定与利福平耐药突变基因检测的新方法

An Innovative Method for Identificatng Mycobacterium Tuberculosis and Indicating It's Rifampin Resistance Simultaneously

目的建立一种可同时进行结核分枝杆菌鉴定并检测结核分枝杆菌利福平耐药突变基因的分子方法。方法以"TGTTCTTCAAGGAGAAGCG"、"TCGTCGGCGGTCAGGTA"为上下游引物对101株结核分枝杆菌复合群及52株非结核分枝杆菌临床分离株进行rpo B基因扩增及序列测定,并应用BLAST数据库和DNAMAN软件进行菌种与rpo B基因突变分析。结果在153例分枝杆菌临床分离株中,104例鉴定为结核分枝杆菌复合群,49例被鉴定为非结核分枝杆菌,与16Sr DNA基因序列鉴定方法符合率为96.73%,与传统分枝杆菌菌种鉴定方法符合率为83%;在104例鉴定为结核分枝杆菌复合群菌株中,54例被检测到rpo B基因突变,与比例法检测利福平耐药表型的符合率为96.15%。结论应用本引物进行rpo B基因序列分析可以一次性完成结核分枝杆菌鉴定与利福平耐药突变基因检测工作,有较大的临床应用价值。

Objective To establish a new method for identification of mycobacteria tuberculosis and rpo B gene mutation of rifampicin resistance. Methods A pair of specific primer for amplification of the third hypervariable region and rifampicin resistance determining region in Mycobacterium genome was designed. The rpo B gene segment of 96 mycobacterium tuberculosis complex strains and 57 nontuberculosis mycobacteria was amplified and sequenced used by this specific primer and all of the gene sequences were analyzed by BLAST and DNAMAN software. Results Among the 153 clinical isolates,one hundred and four were identified as mycobacterium tuberculosis complex and 49 as nontuberculosis mycobacteria,including14 mycobacterium intracellular,12 mycobacterium abscessus,4 mycobacterium fortuitum,1 mycobacterium asiaticum,7 mycobacterium colombiense,3 mycobacterium avium,3 mycobacterium parascrofulaceum,1 mycobacterium sinense,1 mycobacterium triplex,1 mycobacterium vulneris and 1 mycobacterium septicum / vulneris. One case could not be identified.Compared with the 16 Sr DNA gene,the accuracy rate of this new method was 96. 73%; Compared with the traditional method,its accuracy rate was 83%. In 104 cases identified as mycobacterium tuberculosis complex,there were 54 cases detected rpo B gene mutation which was determined resistant to RFP while 50 cases had no mutation and determined sensitive to RFP.Compared with traditional drug sensitivity test,the accurate rate of this new method was 96. 15%. Conclusion This new method is very useful because it can be identification of common mycobacterium species and detection the rifampin resistant through rpo B gene sequence analysis by only one test.