【作者】 阴灵芳；

【导师】 蒙世杰；

【作者基本信息】 西北大学 ， 动物学， 2003， 硕士

【摘要】 结核病是严重危害人类健康的一种传染性疾病，而利福平是最主要的抗结核药物之一，它在结核病的短程化治疗中起着重要的作用。近年来，结核分枝杆菌耐药株的出现特别是耐利福平菌株的出现，严重影响了化疗的效果。用传统的方法进行利福平耐药性的检测，因为耗时长，已不能满足短程化疗的需要。据报导，大于96％的结核分枝杆菌临床分离株耐利福平都是由于rpoB基因发生突变所致。目前，针对rpoB基因突变已出现了很多利福平耐药性的快速检测方法。其中，PCR-SSCP技术应用最为普遍。 本研究首先通过聚合酶链反应—单链构象多态性分析技术对37株结核分枝杆菌临床分离株的rpoB基因进行突变检测，目的在于探讨PCR-SSCP作为新的分子药敏试验方法的应用价值。随后通过直接测序法对陕西区域内结核分枝杆菌临床分离株rpoB基因PCR扩增产物进行序列分析，了解该区域rpoB基因突变情况，为研究结核分枝杆菌耐利福平的分子机制提供参考。 PCR-SSCP分析结果表明，以结核分枝杆菌标准株H₃₇Rv为对照，18株耐利福平菌株中有17株rpoB基因SSCP图谱异常，检测的敏感性为94.4％。而在19株利福平敏感株中也有3株rpoB基因SSCP图谱异常，检测的特异性为84％。与DNA测序结果相比较，PCR-SSCP除一个耐药株的rpoB基因突变没有检出外，其它检测结果均与DNA测序结果相一致，这进一步说明PCR-SSCP是一种快速、灵敏的突变检测技术。 此外，DNA直接测序结果显示，18株耐利福平菌株中，一株rpoB基因的513、514位密码子发生了缺失，其它17株的rpoB基因皆发生了单碱基突变。其中8株为531位密码子TCG→TTG的突变，5株为526位密码子CAC→TAC或AAC或CGC的突变，3株为516位密码子GAC→GTC的突变，另一株发生了513位密码子CAA→AAA的突变。同时，在12株利福平敏感株中，3株耐非利福平菌株分别在rpoB基因的531、526、511位密码子发生了与耐利福平有关的突变，可能是药敏结果有误所致。 这些研究结果进一步证实，结核分枝杆菌耐利福平是由于rpoB基因突变所致，采用PCR-SSCP可快速检测结核分枝杆菌对利福平的耐药性。更多还原

【Abstract】 Tuberculosis is an epidemic disease which threats people’s health severely. Rifampin is one of the most important anti-tuberculosis medicines and plays an important role in a short-term chemotherapy for tuberculosis. In recent years, the emergence of drug-resistant Mycobacterium tuberculosis, especially rifampin-resistant Mycobacterium tuberculosis, has brought a bad efect to the chemotherapy result. The traditional testing method for rifampin-resistance can no longer satisfy the requirement of the short-term chemotherapy because it takes a long time. As reported, it is about 96% of rifampin-resistant Mycobacterium tuberculosis clinical isolates which resulted from the rpoB gene mutation, for which, several methods for rapid testing of rifampin-resistance have been developed. Among these methods, PCR-SSCP is a widly applied technology.This research began with detecting the rpoB gene mutations of 37 Mycobacterium tuberculosis clinical isolates by polymerase chain reaction - single strand conformation polymorphism(PCR-SSCP) to study the application value of this method as a new molecule testing method for drug susceptibility. Then, to get more knowledge about the rpoB gene mutations and provide some scientific information to study the molecule mechanism of resistance to rifampin in mycobacterium tuberculosis, the rpoB gene sequence of mycobacterium tuberculosis clinical isolates from shannxi was analysed by direct sequencing of PCR products.The analysis of PCR-SSCP shows that, in contrast to Mycobacterium tuberculosis strain H37Rv, 17 out of 18 rifampin-resistant strains were observed that their rpoB genes were with abnormal SSCP patterns, and the sensitivity in the test is 94.4%. 3 out of 19 rifampin-susceptible isolates were observed that their rpoB genes were with abnormal SSCP patterns, and the specificity in the test is 84%. Compared with the results of DMA direct sequencing, PCR-SSCP has the same results as that except for failing to check out the rpoB gene mutation of a rifampin-resistant isolate. It proves that PCR-SSCP is a rapid and sensitive mutation testing method.In addition, the results of DNA direct sequencing reveal that 1 out of 18 rifampin-resistant isolates has deletion in codons 513 and 514 of rpoB, and the other 17 are all singe-point mutation in their rpoB genes. Among the 17 isolates, 8 isolates display TCG-TTG mutation in codon 531, 5 isolates have CAC-TAC or AAC or CGC mutation in condon 526, 3 isolates have GAC-GTC mutation in codon 516, and the last one has CAA- AAA mutation in codon 513. Meanwhile, among 12 rifampin-susceptible isolates, 3 non-rifampin-resistant isolates have mutation relating rifampin-resistance in codon 531, 526 or 511 of rpoB gene respectively. It probably resulted from the incorrect result of drug susceptibility testing.The research results further prove that resistance to rifampin in Mycobacterium tuberculosis resulted from the rpoB gene mutation and PCR-SSCP can quickly detect rifampin resistance in Mycobacterium tuberculosis clinical isolates.更多还原