Pengembangan Loop Mediated Isothermal Amplification (LAMP) untuk Deteksi Multidrug Resistant Tuberculosis (MDR-TB) dengan Marker rpoB S450W dan katG S315T

Dian Ritma Setyorini, Prof. dr. Tri Wibawa, Ph.D., Sp.MK(K); Kiki Kurniawan, Ph.D.

2025 | Tesis | S2 Bioteknologi

Indonesia diketahui memiliki jumah kasus infeksi Mycobacterium tuberculosis tertinggi ke-2 di dunia setelah India. Bersamaan dengan meningkatnya jumlah kasus tuberkulosis, kemunculan resistensi terhadap obat antibiotik juga semakin tinggi sehingga diperlukan strategi medikasi yang tepat. Sifat resistensi tuberkulosis terhadap antibiotik lini pertama seperti rifampicin dan isoniazid disebut sebagai multidrug resistant tuberculosis (MDR-TB). Resistensi disebabkan oleh mutasi pada coding region berbagai gen pengkode protein fungsional sehingga menyebabkan penurunan afinitas ikatan protein terhadap antibiotik. Single nucleotide polymorphism (SNP) pada gen rpoB, katG, inhA, embB, dan pncA telah banyak diasosiasikan dengan sifat resistensi tinggi sehingga dapat digunakan sebagai biomarker dalam metode diagnostik molekular. Loop mediated isothermal amplification (LAMP) telah banyak digunakan dalam pengembangan deteksi penyakit infeksius, termasuk tuberkulosis. Penggunaan LAMP yang mudah, cepat, akurat, dan terjangkau menjadikannya sebagai kandidat rapid diagnostic tools yang potensial untuk deteksi MDR-TB. Penelitian ini bertujuan untuk mengembangkan set primer LAMP yang didesain untuk mengamplifikasi marker potensial MDR-TB. Identifikasi genotipik mutasi menggunakan sanger sequencing pada sampel DNA tuberkulosis asal Padang dan Yogyakarta menggunakan 5 gen target. Optimasi LAMP dilakukan secara colorimetric dan real-time, sedangkan performa pengujian ditinjau berdasarkan limit of detection (LOD). Hasil menunjukkan 2 marker potensial yang dapat digunakan sebagai set primer LAMP, yaitu S450W rpoB (RM2) dan S315T katG (RM4). Limit deteksi S450W rpoB colorimetric pada 58.100 copy/test dan real-time pada 581 copy/test. Limit deteksi S315T katG colorimetric pada 539.900copy/test, sedangkan real-time pada 53.990 copy/test. Threshold G/R ratio untuk colorimetric LAMP ditentukan pada nilai 0,7 untuk diferensiasi hasil positif dan negatif.

Indonesia is the 2nd highest number of Mycobacterium tuberculosis infection cases  in the world after India. Along with the increasing number of tuberculosis cases, the emergence of resistance against antibiotic drugs is also getting higher, indicates the appropriate medication strategies are needed. The resistant traits of tuberculosis against first-line antibiotics such as rifampicin and isoniazid is referred to as multi drug resistant tuberculosis (MDR-TB). Resistance is caused by mutations in  the coding regions  of various functional protein-coding genes that lead to a decrease of protein-antibiotic biding affinity. Single nucleotide polymorphism (SNP) in rpoB, katG, inhA, embB, and pncA  gene has been known to be associated with high resistance properties, indicateds its potential as biomarker in molecular diagnostic methods. Loop mediated isothermal amplification (LAMP) has been widely used as detection tools of several infectious diseases, including tuberculosis. The easy, fast, accurate, and affordable use of LAMP makes it a potential candidate for  rapid diagnostic tools for MDR-TB detection. This study aims to develop LAMP primers designed to amplify potential markers of MDR-TB. Genotypic identification of mutations conducted by sanger sequencing on clinical DNA samples from Padang and Yogyakarta using 5 target genes. LAMP optimization include colorimetric and real-time, while test performance is reviewed based on limit of detection (LOD). The results showed 2 potential markers that could be used as LAMP primer sets, S450W rpoB (RM2) and S315T katG (RM4). The detection limit of the S450W rpoB colorimetric is 58,100 copy/test and real-time at 581 copy/test. The detection limit of the S315T katG colorimetric is 539,900 copy/test, while the real-time is at 53,990 copy/test. The threshold value for G/R ratio in colorimetric LAMP is determined at a value of 0.7 for the differentiation of positive and negative results.

Kata Kunci : Mycobacterium tuberculosis, loop mediated isothermal amplification, rpoB, katG, limit deteksi