Pustaka Ilmiah Universitas Padjadjaran

Proceeding Biotech

Detection Of Isoniazid Resistance Mycobacterium Tuberculosis Strains By Multiplex Allele-specific Pcr From Indonesia

Tuberculosis (TB) is an infected diseased caused by Mycobacterium tuberculosis, and treatment with anti-tuberculosis drugs could cured the disease. Multi-drug resistant TB (MDR-TB)has been define by World Health Organization asstrains of tuberculosis that are resistant to at least the two main first-line drugs,namely isoniazid (INH) and rifampicin (RIF). One particular substitution reported to be the most frequent that confers resistance to INH is G944C, in codon 315 which replaced, AGC to ACC, henceSer mutated toThr. Our research group has six clinical isolates MDR-TB, which genotypically showedmutation in rpoB gene codon 526 and 531, but no mutation found in katG315 based on PCR experiment assays. The aim of our research is to find the genotype information of the aboveresistance INH in six clinical isolates. Our research methods consist of PCR multiplex specific alleles katG assays, gel agarose electrophoresis, nucleotidessequencings, and in silico analysis. The PCR multiplex asay employed three primers, two outerprimers KF and KR; and one inner reverse primer K315. Here we showed that multiplex PCR of all isolates showed DNA fragment of 0.43kb and 0.29kb bands. Electrophoregram of 0.43kb katG gene fragment werecompared to the same fragment of M. tuberculosis wild type. Homology analysis showed three isolates have mutation in nucleotide 946, G to T; and an isolate (R2) has mutation in nucleotide 869, C to T. Two other isolates, L4 has mutation in nucleotide 795, G to A; and L7 isolate have two mutations in nucleotides 944 and 946 which changeG to C and G to T respectively. Pymol modeling showed each amino acid position in three dimensions structure protein as the effect of mutation in the DNA level. Data analysis obtained here showed that G946T mutation in three isolate located in codon 316, GGC change to TGC, with the consequence of replacingamino acid glycine to cysteine. Pymol program showed the three dimensional structure of catalase-peroxidase enzyme, and the residue 316 was located closed tothe active site of the enzyme. The recent report have proved that mutation in theresidue 315 confers INH resistance. The fact that three isolates have no mutation in the residue 315, we suggest thatmutation in residue 316 was responsible for the resistance phenotype. The R2 isolate hasmutation in nucleotide C869T located in codon 290, GCT change to GTT, hence amino acid alanine replaced by valine. Pymol program showed amino acid residue 290 located in theloop region of N terminal and was located far from the active site. The effect of this mutation and the relation in resistance INH has not been yet known. L4 isolate hasmutation in nucleotide G795A located in codon 265, TTG change to TTA, which is asynonym substitution.Therefore, the caused of resistance of INH in the L4 isolate have not been yet known. While L7 isolate have mutation in codon 315, which is confer INH resistance. The implication of our results is to give the new information about mutation position in katG gene M. tuberculosis which is resistance INH becaues of mutaiton G946T (glycine316cysteine) in isolate L10, L18, L19; and C869T (alanin290valin) in isolate R2 have not been published before.