[运用反向斑点杂交法检测耐利福平结核分枝杆菌的rpoB基因突变]

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

作者信息

Wang Wen, Pan Wei, Jin Weirong, Weng Xinhua, Yan Ran, Su Bei, Chen Shu, Zhang Wenhong, Lu Hongzhou, Qi Zhongtian

机构信息

Department of Microbiology, Second Military Medical University, Shanghai 200433, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2002 Oct;25(10):591-4.

PMID:12490124

Abstract

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.

摘要

目的

建立一种简便、快速且灵敏的杂交方法，用于检测结核分枝杆菌中与耐药相关的基因突变。

方法

设计14条用于检测结核分枝杆菌中rpoB基因突变型和/或野生型的单链特异性探针，点样并固定于尼龙膜上，采用生物素标记的下游引物获得生物素标记的PCR产物，然后与链霉亲和素 - HRP及TMB进行杂交和分析。

结果

分析了23株耐利福平结核分枝杆菌分离株和11株利福平敏感分离株。分别在30/34和28/34的分离株中，基因突变结果与DNA测序及体外药敏试验结果一致。23株耐利福平分离株中有11株存在Ser - 531突变，其次是His - 526、Leu - 533，13株分离株（包括2株耐利福平分离株）未发现突变。未检测到516和513位点的突变。