A PCR Based Reverse-Line Blot Hybridization Method For Rapid Detection Of RpoB Mutations In Isolates Of Mycobacterium Tuberculosis

Objective To establish PCR based oligonucleotide probe reverse-line blot hybridization(RLBH) for rapid detection of rpoB gene mutations in "hot mutation region" in mycobacterium tuberculosis; to evaluate whether it can be used as susceptibility screening of M. tuberculosis to rifampin or not; and to assess its value in clinical applications.Methods Mutation in the rpoB gene associated with rifampin resistance were tested in 62 rifampin-resistant and 40 rifampin-sensitive clinical strains of Mycobacterium tuberculosis by DNA sequencing and the PCR based reverse-line blot hybridization assay. The results of the two method were compared to evaluate the accuracy rate of reverse-line blot hybridization assay. The rate of concordance of the results of reverse-line blot hybridization assay with the results of susceptibility test was also assessed.Results No mutation was found in 40 rifampin-sensitive strains by both DNA sequencing and the PCR based reverse-line blot hybridization assay. Mutations were found in 55 of 62 rifampin-resistant isolates by DNA sequencing, the mutation rate was 88.7% (55/62). Mutations were found in 54 of 62 rifampin-resistant isolates by reverse-line blot hybridization assay. The sensitivity of this method is 87.1% (54/62), positive predictive value is 100%, without false positive results. All rifampin-sensitive strains were identified correctly, so the specificity is 100%, negative predictive value was 83.3%(40/48). The rate of concordance of the results of the PCR based reverse-line blot hybridization assay with the results of susceptibility test was92.2 % (94/102). Conclusion Both sensitivity and specificity of PCR based oligonucleotide probereverse-line blot hybridization assay are very high; furthermore , there are no falsepositive result, so it is useful in susceptibility screening of M. tuberculosis torifampin.