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[Detecting rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis by using the reverse dot-blot
Wen Wang1, Wei Pan, Weirong Jin
1Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
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.