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Indian J Med Res. 2019 Jul;150(1):33-42. doi: 10.4103/ijmr.IJMR_374_18.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay.

Author information

Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.
Department of TB & Respiratory Diseases, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.


Background & objectives:

Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens.


Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST).


The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR).

Interpretation & conclusions:

The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.


Drug susceptibility testing; multiple allele-specific PCR; nested allele-specific PCR; nested multiple allele-specific PCR; rapid diagnosis

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