论文部分内容阅读
目的利用一种新型的反向点杂交膜片,快速检测结核分枝杆菌耐利福平rpoB基因突变。方法根据结核分枝杆菌rpoB基因序列设计寡核苷酸探针并固定于尼龙膜上,用生物素标记的引物增扩结核分枝杆菌rpoB基因片断,与膜片上探针杂交,膜片检测结果与常规药敏试验结果和测序结果进行对照。结果对63株临床分离株进行检测,10株利福平敏感株未检测到突变,53株耐利福平株,其中48株检测到突变。灵敏度为90.6%,特异度为100%,阳性预测值100%,阴性预测值66.7%。用反向点杂交膜片检测27份肺结核患者的痰标本,17份非结核的痰液标本。12份耐利福平的痰标本中,11份检测到突变,膜片检测与药敏结果的符合率为92.3%;15份利福平敏感的痰标本中,膜片检测结果有6份出现突变信号;17份非结核病的其他患者的痰液标本中有1份检测到突变。检测痰标本的灵敏度为91.7%,特异度为78.1%,阳性预测值61.1%,阴性预测值96.1%。结论反向点杂交膜片可简便、快速、较准确地检测出大多数结核分枝杆菌耐利福平分离株的rpoB基因突变。
Objective To rapidly detect the rpoB gene mutation of rifampicin-resistant Mycobacterium tuberculosis using a new type of reverse dot blot hybridization membrane. Methods According to the sequence of rpoB gene of Mycobacterium tuberculosis, oligonucleotide probes were designed and immobilized on nylon membrane. The fragments of rpoB gene of Mycobacterium tuberculosis were amplified by biotin-labeled primers and hybridized with probes on the membrane. Results were compared with conventional susceptibility and sequencing results. Results Sixty-three clinical isolates were tested. No mutation was detected in 10 rifampin-susceptible strains, and 53 were resistant to rifampicin. 48 of them were detected mutations. Sensitivity was 90.6%, specificity was 100%, positive predictive value was 100%, negative predictive value was 66.7%. The sputum specimens from 27 patients with pulmonary tuberculosis and 17 specimens of non-tuberculosis sputum were detected by reverse dot blot hybridization. Of 12 sputum samples with rifampicin, 11 were detected mutations, the coincidence rate of patch detection and drug susceptibility results was 92.3%. Six of the 15 sputum samples with rifampin sensitivity were detected in the sputum samples Mutation signal; 1 out of 17 sputum specimens from other non-TB patients detected mutations. The sensitivity of sputum samples was 91.7%, the specificity was 78.1%, the positive predictive value was 61.1% and the negative predictive value was 96.1%. Conclusion Reverse dot blot hybridization can detect the rpoB gene mutation of most M. tuberculosis rifampicin isolates easily, rapidly and accurately.