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

• Published in: Zhonghua jiehe he huxi zazhi Vol. 25; no. 10; p. 591
• Main Authors: Wang, Wen, Pan, Wei, Jin, Weirong, Weng, Xinhua, Yan, Ran, Su, Bei, Chen, Shu, Zhang, Wenhong, Lu, Hongzhou, Qi, Zhongtian
• Format: Journal Article
• Language: Chinese
• Published: China 01.10.2002
• Subjects: Antibiotics, Antitubercular - pharmacology; DNA, Bacterial - chemistry; DNA-Directed RNA Polymerases - genetics; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis - drug effects; Mycobacterium tuberculosis - genetics; Nucleic Acid Hybridization; Rifampin - pharmacology
• ISSN: 1001-0939

To establish a simple, rapid and sensitive hybridization method for detecting drug-resistance relevant gene mutation in Mycobacterium tuberculosis. 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. 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. Reverse dot-blot hybridization may be of potential