Detection of mutations in drug resistance genes of Mycobacterium tuberculosis by a dot-blot hybridization strategy

Abstract

Setting Mycobacterium tuberculosis isolates from patients in communities endemic for tuberculosis in South Africa.Objective To develop a reliable PCR-based dot-blot hybridization strategy to detect mutations conferring drug resistance.Design Different loci in six genes associated with drug resistance to isoniazid, rifampacin, streptomycin and ethambutol were selected to develop the PCR-based dot-blot hybridization strategy.Results Primers and probes to detect mutations at codons 315, 463 (kat G) 269 (kas A), 531, 526 (rpo B) 43 (rps L), 513 (rrs) and 306 (emb B) were designed and used to develop a PCR-based dot-blot hybridization strategy. The dot-blot hybridization strategy with wild-type probes can efficiently be used to detect drug resistant mutations since these do not hybridize to mutant loci. Stripped blots and mutant probes can be used to identify the precise mutation. The emb B gene (ethambutol resistance) was used to show how the dot-blot strategy can assist with the prediction of drug resistance more accurately. The method is rapid, reproducible, not technically demanding and samples can be done in batches. Additional loci can easily be incorporated.Conclusions A PCR-based dot-blot hybridization strategy is described which can accurately identify drug resistant strains and the method is useful for patients at risk and in areas endemic for tuberculosis.