应用反向线性杂交技术快速检测结核分枝杆菌利福平耐药性研究

Rapid Detection of Rifampin-resistant Mycobacterium Tuberculosis Strains Using Reverse Line Blot Assay

探讨反向线性杂交技术(reverse line blot assay,RLB)在结核分枝杆菌利福平(RFP)耐药性快速检测中的应用价值。采用RLB将包含rpoB基因核心区的扩增产物与标记特异性探针的膜进行杂交,共对121株结核分枝杆菌进行RFP耐药性检测,并将结果与常规药敏实验进行比较。结果发现,121株中有71株对RFP耐药,56株为多重耐药株;采用RLB共检测到65株RFP耐药株rpoB基因核心区存在突变,其灵敏度为91.5%(65/71);50株RFP敏感株中均未检测到突变,则特异性为100%(51/51);56株多耐药菌株中,92.9%(52/56)存在rpoB基因核心区突变。因此,用RLB检测结核分枝杆菌RFP耐药株具有快速,高效,特异性和灵敏度高的优点,且可用于多耐药菌株的筛选,具有推广和潜在的临床应用价值。

To evaluate the reverse line blot assay（RLB） in the detection of rifampin （RFP）-resistant Mycobacterium tuberculosis （MTB） strains. The amplified PCR fragments containing hot-spot region of rpoB gene were used to hybridize with specific probes labeled membrane by using RLB. Total 121 strains of MTB were analyzed, and the results were compared with standard drug susceptibility tests of MTB. Of the total 121 isolates, 71 were resistant to RFP, while 56 were multi-drug resistant （MDR） strains. Sixty-five RFP resistant strains were found to harbor mutations in the rpoB hot-spot region by RLB. No mutation was detected in RFP sensitive strains. The sensitivity and specificity of RLB to detect RFP resistant strains were 91.5% （65/71） and 100% （50/50）, respectively. Interestingly, 52 （92.9%） of 56 MDR strains harbor mutations in rpoB hot-spot region. The RLB was a rapid, simple, sensitive and highly specific method to detect RFP resistant and MDR strains, It could be a practical clinical tool in routine diagnostic work.