逆向点杂交方法检测耐利福平结核分支杆菌rpoB基因突变

目的 建立一种灵敏、特异、快速检测结核分支杆菌耐药相关基因突变的方法，用于临床对利福平耐药的快速诊断。方法 将14条特异性探针固定在尼龙膜上，通过在下游引物标记生物素的方法得到生物素标记的结核分支杆菌DNA PCR扩增产物，与固定在尼龙膜上的特异性探针杂交，杂交物通过链酶亲和素标记辣根过氧化物酶及底物（四甲基联苯胺）显色判定结果。将杂交结果与基因测序结果及药敏试验结果进行对比分析。结果 采用逆向点杂交法共检测23株耐利福平及11株敏感型菌株，与药敏试验结果、测序结果符合率分别为28／34和30／34。所发现突变类型依次为：第531位突变11株，第526位突变7株，第533位突变3株。未检测到第516位、第513位碱基突变。结论 该方法可用于临床结核分支杆菌对利福平耐药性的快速检测。

Detecting rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis by using the reverse dot-blot hybridization method

Objective To establish a simple, rapid and sensitive hybridization method for detecting drug resistance relevant gene mutation in Mycobacterium tuberculosis Methods Fourteen single strand specific probes designed to detect mutated and / or wild rpoB gene in Mycobacterium tuberculosis were spotted and fixed on nylon membranes, and PCR products labeled with biotin were obtained by using down stream primer labeled with biotin, then hybridized and analyzed with streptavidin HRP and TMB Results Twenty three rifampin resistant Mycobacterium tuberculosis isolates and 11 rifampin sensitive isolates were analyzed. The gene mutations were consistent with the DNA sequencing and the in vitro susceptibility test in 30/34 and 28/34 of the isolates, respectively. Mutations of Ser 531 were present in 11 of the 23 rifampin resistant isolates, followed by His 526, Leu 533, and no mutation was found in 13 isolates, including 2 rifampin resistant isolates. Mutations in loci 516 and 513 were not detected Conclusion Reverse dot blot hybridization may be of potential use for the rapid diagnosis of rifampin resistant tuberculosis.