Primer design and optimization of loop-mediated isothermal amplification (LAMP) for specific detection of genes isoniazid-resistant Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains one of the leading causes of death in Indonesia. The limited diagnostic tools for TB, including Multi-Drug Resistant Tuberculosis (MDR-TB), contribute to the increasing number of cases. MDR-TB refers to TB strains resistant to Anti-Tuberculosis Drugs such as Isoniazid (INH). In response, the WHO has recommended the use of a rapid, equipment-free method called loop-mediated isothermal amplification (LAMP). This study aims to design primers and optimize the LAMP method for the detection of Isoniazid-resistant M. tuberculosis. The research integrates bioinformatics testing, laboratory experiments, and validation methods. The study successfully designed LAMP primers for detecting Isoniazid-resistant comprising three pairs of primers: F3, B3; FIP, BIP; and LF, LB. The optimization of the LAMP method involved temperature and time parameters. Five temperature treatments were tested, revealing an optimum temperature of 70°C. For time optimization, eight treatments were conducted, with an optimum time range of 25-30 minutes. The study concludes that the LAMP method differs from the PCR method due to the higher number of primers employed and LAMP requires only a single isothermal temperature. Additionally, the study achieved optimization of temperature and time for the LAMP method, facilitating the detection of Isoniazid-resistant MTB.