Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization

Biomed Environ Sci. 2015 Jan;28(1):25-35. doi: 10.3967/bes2015.003.

Abstract

Objective: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB 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.

Keywords: Mycobacterium tuberculosis; Reverse dot blot hybridization; Rifampin-resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antitubercular Agents / pharmacology*
  • Drug Resistance, Bacterial*
  • Genotype
  • Immunoblotting / methods*
  • Microbial Sensitivity Tests
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / genetics
  • Polymerase Chain Reaction / methods
  • Rifampin / pharmacology*
  • Sensitivity and Specificity
  • Time Factors

Substances

  • Antitubercular Agents
  • Rifampin