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


CONCLUSIONS
Reverse dot-blot hybridization may be of potential use for the rapid diagnosis of rifampin-resistant tuberculosis.
Wang
Wen
W
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Pan
Wei
W
Jin
Weirong
W
Weng
Xinhua
X
Yan
Ran
R
Su
Bei
B
Chen
Shu
S
Zhang
Wenhong
W
Lu
Hongzhou
H
Qi
Zhongtian
Z
D000904:Antibiotics, Antitubercular / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D012293:Rifampin / Q000494:pharmacology*
D000904:Antibiotics, Antitubercular; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin