PCR-SSCP技术快速检测结核分枝杆菌rpoB基因突变的研究

目的 应用PCR-SSCP技术直接快速检测痰标本中结核分枝杆菌rpoB基因突变，评价此方法用于结核分枝杆菌利福平 (RFP)耐药性的意义。方法 以结核分枝杆菌H37Rv为对照、50例耐RFP(单耐和多耐)结核分枝杆菌临床分离株、12例RFP敏感菌株、35例耐RFP肺结核患者痰标本，采用PCR-SSCP检测痰标本和菌株中结核杆菌rpoB基因突变。结果 50例耐RFP菌株中有45例PCR-SSCP条带与结核分枝杆菌H37Rv条带有明显差别，与药敏试验结果做对比，PCR-SSCP检测敏感性为90%，特异性为100%；12例敏感菌株与H37Rv条带一致，35份肺结核病人痰标本，21份痰PCR扩增阳性，15份SSCP图谱与H37Rv图谱有差别，而且PCR-SSCP28份图谱与H37Rv有差别，阳性率为80%。结论 PCR-SSCP检测结核分枝杆菌rpoB基因突变快速、简便、易行，有望用于结核分枝杆菌RFP耐药性的快速测定，并将成为直接检测临床标本中结核分枝杆菌耐RFP快速方法。

Rapid technique in detection of rpoB gene mutation in Mycobacterium tuberculosis by PCR-SSCP

Objective To evaluate rifampin-resistant Mycobacterium tuberculosis by PCR-SSCP technique to detect rpoB gene mutation in Mycobacterium tuberculosis of sputums. Methods Fifty clinical isolates of rifampin-resistant and multidrug resistant Mycobacterium tuberculosis, twelve sensitive strains, Hiirty-five rifampin-resistant strains from sputum specimens of patients with pulmonary tuberculosis, were analyzed by PCR-SSCP technique, with H₃₇Rv reference as control group. Results The characteristics of PCR-SSCP grah in fifty strains of rifampin-resistant were different from H₃₇Rv except for five strains. In comparison with susceptibility testing methods,the sensitivity and specificity were 90% and 100% respectively. Twelve sensitive strains were same as H₃₇Rv. In thirty-five strain from sputum specimens of patients with pulmonary tuberculosis,twenty-one strains of them were positive by PCR. The characteristic of SSCP graph in fifteen of them were different bom H₃₇Rv, and the characteristics of PCR-SSCP graph in twenty-eight of them were different from H₃₇Rv, the positivity was 80%. Ccmdusicm llie PCR-SSCP can easily and rapidly detect rifampin resistance of Mycobacterium tuberculosis,the technigue might be used for rapidly detect Mycobacterium tuberculosis rifampin resistance in clinical specimen directly.