Detection of rifampicin resistance in sputum samples by PCR-ELISA

ABSTRACT Introduction Tuberculosis caused by Mycobacterium tuberculosis shows resistance to anti-tuberculosis drugs like Rifampicin and Isoniazid generating a public health problem. Using molecular techniques, such as PCR-ELISA, is an alternative that will detect resistance to RIF easily and economically. Objective Optimize a PCR-ELISA that can detect resistance to RIF in sputum samples. Method A PCR-ELISA was standardized to amplify a 255bp of rpoB gene that encodes resistance to RIF. Different parameters were optimized: hybridization temperatures, and probe concentration, among others. The technique’s analytical sensitivity, specificity, and concordance against GenotypeMTBDRplus v2 were evaluated using 20 samples in a pilot assay. Results A PCR-ELISA was optimized for the detection of resistance mutations; the analytical sensitivity of the PCR-ELISA was 4ng of PCR product for the H445D and H445Y probes while for the S450L probe the sensitivity was 0.4 ng of PCR product respectively. The technique has a specificity of 100%. Two mutations, S450W and L452P, were not detected by our system. In the pilot assay, a “good agreement” (k=0.737) was obtained between both techniques. Conclusions A PCR-ELISA was standardized for the detection of the 3 more frequent mutations in Peru associated with resistance to RIF simultaneously, the most frequent being S450L.