Detection of resistance genes of rifampin and isoniazid in Mycobacterium tuberculosis by using gene chips
Objective To establish a method for detection of the resistance genes in Mycobacterium tuberculosis by genes chips. Methods Designed four primers characterized by digoxin, four gene fragments of rpoB, katG, inhA and ahpC in Mycobacterium tuberculosis were amplified. Probes were designed and the gene chip was fabricated according to the 25 single nucleotide polymorphisms of 8 sites on 4 genes associated with RFP and INH resistance, amplified products were hybridized with chips. Results Four kinds of amplification products were identified and contain 165, 181, 245 and 315 bp respectively. The method may detect the signals of wildness and mutation in every sites even when the concentration of Mycobacterium tuberculosis was 1.6×103/ml. The amplified products of 19 nan-Mycobacterium tuberculosis standard strains and 9 non- Mycobacterium standard strains didn't have signals when hybridized with chips. Every site of standard strain of H37Rv Mycobacterium tuberculosis was wildness. Five clinical isolates of Mycobacterium tuberculosis were detected five times repeatedly, the results were correspondent. In 46 clinical isolates, 28 strains were resisted to RFP, in which 24 strains were mutated, and the percent of mutation was 85.7% ; Eighteen strains were sensitive to RFP, 2 strains were mutated; Thirty-one strains were resisted to INH and 20 strains were mutated, the percent of mutation was 64.5% ; Fifteen strains were sensitive to INH, 4 strains were mutated. Conclusion Coloration gene chip for detecting resistance gene in Mycobacterium tuberculosis is highly sensitive and specific, and is technically feasible for clinical application.