反相斑点杂交快速检测结核分枝杆菌rpoB基因突变

Rapid detection of rpoB gene mutations of rifampin resistance in Mycobacterium tuberculosis by PCR based reverse-line blot hybridization

目的建立以聚合酶链反应(PCR)为基础的寡核甘酸探针反相斑点杂交方法快速检测对利福平耐药的结核菌相关的rpoB基因突变,并评价其临床应用价值。方法应用PCR反相斑点杂交方法对利福平耐药的62株结核菌和对其敏感的40株结核菌进行检测,所得结果与传统药敏试验结果以及102株结核菌的直接测序结果进行比较。结果62株对利福平耐药的菌株中,54株碱基突变被准确鉴定,灵敏度为87.1%(54/62),阳性预测值为100%;40株对利福平敏感的菌株均被准确鉴定,特异性为100%,阴性预测值为83.3%(40/48);准确度为92.2%(94/102)。与测序法结果符合率为99%。结论以PCR为基础的反相斑点杂交方法其敏感度和特异性均高,无假阳性结果,因此适用于大批量结核菌对利福平耐药性的初筛。

Objective By use PCR based oligonucleotide probe reverse-line blot hybridization to detect rpoB gene mutations of rifampin resistance mycobacterium tuberculosis, and to assess its value in clinical applications.Methods A total of 102 M. tuberculosis isolates were tested by both the PCR based reverse-line blot hybridization assay and direct sequencing .The results were compared.Results 54 of 62 rifampin-resistant isolates′ mutations were correctly identified(sensitivity 87.1%, positive predictive value 100%). All of the 40 rifampin-susceptible isolates were correctly identified(specificity 100%, negative predictive value was 83.3%).Compared with the direct DNA sequencing results, the accuracy rate of reverse-line blot hybridization was 99%.Conclusion Both sensitivity and specificity of PCR based oligonucleotide probe reverse-line blot hybridization assay were very high. There was no false positive result exists. It was useful for rapid primary screening for rifampin resistance strains of M. tuberculosis.