Yin Ling Fang, Lu Xue Dong, Meng Shi Jie, Wang Rui
College of Life Science, Northwest University, Xi'an.
Fen Zi Xi Bao Sheng Wu Xue Bao. 2006 Apr;39(2):185-9.
ABSTRACT With Mycobacterium tuberculosis H37Rv as a control, the mutations of rpoB gene from 37 Mycobacterium tuberculosis clinical isolates (18 rifampin-resistant strains and 19 rifampin-susceptible strains) have been detected by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) under standard condition (no glycerol in gel). For the strains in which mutations had not been detected under standard condition, PCR-SSCP analysis was performed again on condition that 8% glycerol was added to gel. In addition, the PCR products of rpoB genes of 30 strains (18 rifampin-resistant strains and 12 rifampin-susceptible strains) were detected by DNA sequencing. After twice analyses of PCR-SSCP, in 18 rifampin-resistant strains, the patterns of 17 strains were found to be abnormal; and in 19 rifampin-susceptible strains, the patterns of 16 strains were found to be normal. Compared with routine drug susceptibility test, the sensitivity of PCR-SSCP 94.4% is higher for rifampin resistance detection, and specificity of PCR -SSCP 84% seems lower. The results of sequence analysis were shown as: amomg 18 rifampin-resistant strains, only one has deletion in codons 513 and 514 of rpoB gene, which is a new report, the others have point mutation; among 12 rifampin- susceptible strains, 3 positive strains in SSCP occur gene mutations directly related to rifampin resistance. Then compared with DNA sequencing, the accuracy, sensitivity and specificity of PCR-SSCP are 96.7%, 95.2% and 100% respectively. Therefore, the sensitivity of mutation detection in SSCP could be improved by adding glycerol in gel. It is feasible and efficient to detect the mutation of rpoB gene in Mycobacterium tuberculosis by PCR-SSCP. This method is valuable to evaluate the rifampin resistance of Mycobacterium tuberculosis in clinical application for curing tuberculosis.
摘要 以结核分枝杆菌H37Rv为对照，采用聚合酶链反应-单链构象多态性分析（PCR-SSCP）方法，在标准条件下（凝胶中不加甘油）检测37株结核分枝杆菌临床分离株（18株耐利福平菌株和19株利福平敏感菌株）rpoB基因的突变情况。对于在标准条件下未检测到突变的菌株，在凝胶中添加8%甘油的条件下再次进行PCR-SSCP分析。此外，对30株菌株（18株耐利福平菌株和12株利福平敏感菌株）的rpoB基因PCR产物进行DNA测序。经过两次PCR-SSCP分析，18株耐利福平菌株中，17株的条带模式异常；19株利福平敏感菌株中，16株的条带模式正常。与常规药敏试验相比，PCR-SSCP检测利福平耐药的敏感性为94.4%，较高，但特异性为84%，似乎较低。测序结果显示：18株耐利福平菌株中，仅1株rpoB基因第513和514密码子有缺失（此为新报道），其余为点突变；12株利福平敏感菌株中，SSCP检测为阳性的3株发生了与利福平耐药直接相关的基因突变。与DNA测序相比，PCR-SSCP的准确性、敏感性和特异性分别为96.7%、95.2%和100%。因此，在凝胶中添加甘油可提高SSCP突变检测的敏感性。采用PCR-SSCP检测结核分枝杆菌rpoB基因的突变是可行且高效的。该方法在临床治疗结核病中评估结核分枝杆菌对利福平的耐药性方面具有重要价值。
