Rapid Detection of Isoniazid and Rifampicin Resistance in Mycobacterium tuberculosis Strains Using Allele-specific PCR

Abstract

Background: Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Rapid molecular diagnosis of tuberculosis in clinical specimens facilitates rapid detection of Mycobacterium tuberculosis. Aim: To develop a nested allele-specific polymerase chain reaction (NAS-PCR) detect isoniazid (INH) and rifampin (RIF) resistance-associated mutations directly from clinical specimens. Materials and Methods- We investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 395 microbiologically confirmed (smear and/or culture) clinical specimens by nested allele-specific PCR assay. Genotypic detection of MTB strains was performed using different primers detecting mutations in codons rpoB516, 526 and 531 and katG 315 and mabA-inhA (-15) promoter region. Results- Two seventeen mutations were detected by NAS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 198 mutations in the katG gene codon 315 and 30 mutations at position -15 in the inhA promoter region that confers resistance to isoniazid. Conclusion- The results of this study suggested that NAS-PCR assay is rapid, cost effective, and easy to perform DNA-based protocol for the detection of RIF and INH resistance M. tuberculosis strains directly from clinical specimens. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of clinical samples.