套式PCR及测序方法用于痰标本中结核分枝杆菌rpoB耐药基因突变检测

目的应用套式PCR-DNA测序方法直接检测痰标本中结核分枝杆菌相关的rpoB基因突变,以期建立一种直接检测分枝杆菌耐利福平的快速方法,并评价其临床应用价值。方法采用套式PCR-DNA测序方法直接检测112例活动性肺结核患者和20例非结核性肺部疾病患者痰标本中结核分枝杆菌rpoB基因突变情况。同份痰标本同时做涂片抗酸染色,罗氏培养及菌型鉴定。结果112例活动性肺结核患者痰标本套式PCR扩增87例呈阳性,产物DNA测序31例有rpoB基因突变,其中分离出耐利福平株的32例痰中29例发生了基因突变,耐药突变率90.6%(29/32),39例菌阴(涂阴培阴)痰中有2例发生突变。分离出对利福平敏感株的37例痰中未发生突变,20例非结核性肺部疾病患者痰标本套式PCR扩增均为阴性,特异性100%。结论套式PCR-DNA测序可望为直接检测临床痰标本中结核分枝杆菌耐利福平的准确、特异、快速的方法。

Detection of rpoB gene mutation of Mycobacterium tuberculosis in sputum specimens by nested PCR- DNA sequencing

Objective To detect rpoB gene mutation of rifampin-resistant Mycobacterium tuberculosis insputum specimens by nested PCR-DNA sequencing,and to evaluate its value in clinical applications to develop arapid method for the detection of rifampin-resistance in Mycobacterium tuberculosis.Method One handred and twelve sputumspecimens from the patients with active pulmonary tuberculosis and 20 sputum specimens from the patients withnon-tuberculous pulmonary disease were detected by nested PCR-DNA sequencing,smear,culture,and speciesidentification.Results Thirty-one of 112 sputum specimens from the patients with active pulmonarytuberculosis had mutations of rpoB gene in M.tuberculosis by nested PCR-DNA sequencing.Of 32 sputumspecimens with rifampin-resistant strains,29 had rpoB mutations,and the mutation rate was 90.6%(29/32).2 of 39 sputum specimens with smear and culture negative had rpoB mutations.No mutation was found in 37 sputumspecimens with rifampin-sensitive strains(specificity 100%). Conclusion Nested PCR-DNA sequencing maybecome an accuracy,specific,rapid method for direct detection of rpoB gene mutation in Mycobacteriumtuberculosis from clinical specimens.