DNA Probe Design for Detection Mutation at Codon 315 In katG Gene of Mycobacterium Tuberculosis to Real-Time Polymerase Chain Reaction

  • I Gusti A. A. Santhi Rahmaryani Udayana University
  • Ni Kadek Ariani Udayana University
  • Dyah Subadrika Warma Dewi Udayana University
  • Ni Komang Sasi Ani Udayana University
  • Ade Ari Sundari Udayana University
  • Kadek Widya Yuli Hartati Udayana University
  • Sagung Chandra Yowani Udayana University
DOI: https://doi.org/10.24843/JHSM.2017.v01.i02.p08

Abstract

High-level resistance to isoniazid as a first-line tuberculosis drugs can be caused by mutations in codon 315 katG


Mycobacterium tuberculosis . Mutation at codon 315 is the most frequent mutation with the highest amino acid variation,


compared to other codons in the Mycobacterium tuberculosis katG  gene. Therefore, a specific probe  is required for rapid and


proper detection of mutations at codon 315. In this study, the design of a nucleotide sequence probe with TaqMan labeling was


performed using Clone Manager Suite 6 software . The mutant probe  obtained was analysed in two stages. The initial analysis


is based on the length of the probe  (22-30 bases), Tm (70ºC), %GC (35-65%), not in hairpin  form, dimer  (< 5 bases), runs  and


repeat  (? 4 for base A, T, C, and < 3 for base G). Furthermore the final analysis was carried out with no G base in 2 bases at


the end of the 5’ probe  and the amount of base C ? G.


The study resulted in 260 probe mutants. After the initial analysis, 11 mutant probes were obtained to recognize mutations in


the codon of 315 katG Mycobacterioum tuberculosis  genes. The probe  consists of 2 probes  for the S315T mutation, 6 probes


for S315N mutation, and 3 probes  for S315V mutation. The criteria of the 11 mutant probes  are 22-23 bases long, Tm 70ºC, %


GC 56-63%, 4 dimer , 2 runs , and does not have repeats  and does not form hairpin  at a temperature of 56ºC. Based on the final


analysis, 3 mutant probes  were obtained fulfilling the TaqMan probe labeling criteria, namely K315MT1 for specific detection


of mutation S?T and K315MN5, then K315MN23 for specific detection of mutation S?N.


The conclusion of this study shows that the best mutant nucleotide sequence probes for the detection of mutations at codon of


315 KatG Mycobacterium tuberculosis  genes are 5’-FAM-CC ACC GGC ATC GAG GTC GTA TG-TAMRA-3’; 5’FAM-ATC


ACC AAC GGC ATC GAG GTC G-TAMRA-3’; dan 5’FAM-C ACC AAC GGC ATC GAG GTC GTA T-TAMRA-3’. The


design of the mutant probe according to the TaqMan probe criterion for real-time PCR was obtained by 3 probes from the 11


selected mutant probes in the initial analysis.