银染单链构象多态性技术检测结核杆菌rpoB基因突变

SSCP technique in detection of rpoB gene mutation in rifampin resistant Mycobacterium tuberculosis

目的探讨分子生物学方法在临床标本结核分枝杆菌利福平（ＲＦＰ）耐药性评估中的应用价值。方法设计针对ｒｐｏＢ基因１８３ｂｐ扩增引物，运用聚合酶链反应单链构象多态性分析（ＰＣＲＳＳＣＰ）银染色方法，检测了４５株结核杆菌临床分离株，并对其中部分菌株进一步做了ＤＮＡ序列分析。结果以药敏试验结果为对照，有１６株ＲＦＰ敏感株及２６株ＲＦＰ耐药株经ＳＣＰ方法得到检测，阳性占９３．３％，特异性１００％。对部分菌株１８３ｂｐ片断测序结果显示，ＲＦＰ敏感株无ｒｐｏＢ基因突变，耐药株均发生碱基突变，分别导致编码５３１及５２６位点的氨基酸改变。结论银染ＰＣＲＳＣＰ方法具有简便，快速，准确的特点，提高敏感性，可以对临床标本结核杆菌利福平耐药突变进行鉴定。序列分析证明结核分枝杆菌利福平耐药菌株有ｒｐｏＢ基因突变。

Objective To study the role of molecular biology technique in evaluating rifampin(RFP)resistance in Mycobacterium tuberculosis. Methods Primers were designed for amplifying 183 bp fragment of rpoB gene ,and 45 isolates of Mycobacteriun tuberculosis were analyzed by PCR SSCP.DNA sequencing was also performed in some of the isolates.Results To Compare the susceptibility test, 16 RFP susceptible strains and 26 RFP resistant strains were detected by SSCP. The positivity and specitivity was 93.3% and 100%,respectively. In DNA sequencing of the 183bp fragment of some strains, RFP resistant phenotype of Mycobacterium isolates experienced nucleotide changes which led to substituted aminoacid at Ser 531 and His 526 in the rpoB gene ,while RFP susceptible strains exhibited no mutations in this region. Conclusion PCR SSCP technique is a simple,rapid and accurate method and would be used to evaluate RFP resistance in Mycobacterium tuberculosis after the sensitivity of rpoB gene amplification by PCR is increased. In DNA sequencing, mutations in rpoB gene occur in RFP resistant strains of mycobacterial tuberculosis.