Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

Objective A PCR-reverse dot blot hybridization(RDBH) assay was developed for rapid detection of rpo B gene mutations in ‘hot mutation region’ of Mycobacterium tuberculosis(M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing(DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value(PPV) and negative predictive value(NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531(48.6%), 526(25.4%), 516(8.8%), and 511(6.6%), and the combinative mutation rate was 15(8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.