DNA probe based colorimetric method for detection of rifampicin resistance of Mycobacterium tuberculosis.

Abstract

Rifampicin resistance of Mycobacterium tuberculosis is due to the occurrence of point mutations of the rpoB gene and the site of mutations vary geographically. Commercialized molecular based methods are not able to comprehensively detect rifampicin resistance as they target a limited number of gene mutations which are thought to be common. The aim of the study was to establish a low cost DNA probe based colorimetric method that can be customized for detection of rifampicin resistance of M. tuberculosis. Thus, enzyme-linked oligosorbent assay (ELOSA) was developed for the detection of polymerase chain reaction (PCR) amplified fragments of rpoB gene of M. tuberculosis DNA on microtiter plates. Forty two M. tuberculosis isolates (rifampicin resistant and susceptible isolates identified by agar proportion method) were used for developing and validating the assay. The point mutations of resistant isolates had been previously determined by DNA sequencing. Two fragments of rpoB gene were labeled with digoxigenin by PCR. The amplified products were hybridized with selected allele specific probes for three mutations and its wild types (six probes) which were captured onto streptavidin coated microtiter plates and detected by color development. Both sensitivity and specificity of all probes were ≥96% and there was excellent discrimination (area under the curve (AUC)>0.9) between rifampicin susceptible cases and resistant cases. The probe-based colorimetric assay (PCR-ELOSA) developed in this study showed good agreement with reference mutations that were confirmed by DNA sequencing. In conclusion, PCR-ELOSA is a reliable and economical assay that can be customized for detection of rifampicin resistance.