Optimization of Annealing Temperature and Primer Concentration for Detection of Mycobacterium Tuberculosis Isoniazid Resistance Real-Time PCR Method

Abstract

Indonesia has the second highest number of TB cases in the world according to the WHO report in 2022. Eradication of the National TB Program is difficult due to the presence of Mycobacterium tuberculosis (M. Tb) bacteria that are resistant to various anti-tuberculosis drugs (OAT) known as Multidrug- Resistant Tuberculosis (MDR-TB). Therefor, currently, a nucleic acid-based diagnostic kit has been developed that is designed to directly detect the presence of resistance to INH with mutations of the KatG gene codon 315 (S315T, S315N, S315I, S315R, S315G, R463L) and rifampicin sensitive M. Tb using the Real-Time PCR method. PCR components that are important for development include annealing temperature and primer concentration. This study aims to determine the optimum annealing temperature and primer concentration. The type of research is the Quasi Experiment. The treatment in this study is by adding each primer that varies in concentration, namely 200nM-400nM while varying the annealing temperature with a range of 52-62°C according to the Tm of each primer. The data obtained from this research are Ct values ​​from the target genes S315T, S315N, S315I, S315R, S315G, R463L and M. Tb Rifampicin sensitive.