DESAIN PRIMER DAN ANALISIS SECARA IN SILICO UNTUK AMPLIFIKASI GEN embB Mycobacterium tuberculosis
DOI:
https://doi.org/10.24843/JCHEM.2026.v20.i01.p16Abstract
First-line anti-tuberculosis drugs (OTD) consist of a combination of Isoniazid, Rifampicin, Pyrazinamide, and Ethambutol. Although this regimen is effective, drug resistance, including resistance to Ethambutol, poses a significant challenge in tuberculosis control. Rapid detection of Ethambutol resistance is crucial for determining appropriate treatment regimens and preventing the spread of resistant TB strains. High-frequency resistance to Ethambutol is generally caused by mutations in the embB gene, particularly at mutation hotspots M306V (ATG to GTG) and Q497R (CAG to CGG) found in Ethambutol-resistant Mycobacterium tuberculosis. This study aimed to obtain the best primer pair designed in silico using the Clone Manager Suite 12 program for use in the Real-Time PCR method. Primer design and analysis were conducted in silico, with specificity and sequence similarity assessed using Primer-BLAST against the NCBI database. The DNA template used for primer analysis was obtained from the NCBI database (accession number: MK579314.1), representing the embB gene sequence of M. tuberculosis H37Rv. The results identified an optimal primer pair: forward primer 5'-ATTGCCCAGCTCCTCCTCAG-3' and reverse primer 5'-CGCCGCAACATGATGAACAC-3'. These primers met the criteria for ideal primer design in terms of length, melting temperature (Tm), GC content, stability, and minimal formation of hairpins, repeats, dimers, and runs. Specificity testing confirmed that the primers exclusively recognize M. tuberculosis, indicating their strong potential for rapid and specific detection of Ethambutol resistance in clinical samples.
Keywords: Primer design, embB gene, in silico, M. tuberculosis, Ethambutol Resistance
Abstrak
Obat Anti Tuberkulosis (OAT) lini pertama terdiri dari kombinasi Isoniazid, Rifampisin, Pirazinamid, dan Ethambutol. Meskipun terapi ini efektif, resistensi terhadap obat, termasuk Ethambutol, menjadi tantangan serius dalam pengendalian TB. Oleh karena itu, dibutuhkan deteksi cepat terhadap resistensi Ethambutol untuk regimen pengobatan yang tepat dan mencegah penyebaran tuberkulosis yang resisten dengan cara yang efisien. Resistensi terhadap Ethambutol dengan frekuensi resistensi tinggi, umumnya disebabkan oleh mutasi gen embB. Mutasi yang dianalisis mencakup M306V (perubahan kodon ATG menjadi GTG) dan Q497R (perubahan kodon CAG menjadi CGG), yang telah dilaporkan sebagai hotspot mutasi pada gen embB M. tuberculosis resisten ethambutol. Penelitian ini bertujuan untuk memperoleh sepasang primer terbaik hasil desain secara in silico menggunakan program Clone Manager suite 12 untuk metode Real-Time PCR. Perancangan primer secara in silico dilakukan menggunakan program Clone Manager Suite 12. Primer yang terpilih dianalisis secara in silico untuk menguji spesifisitas dan tingkat kesamaan sekuens pada database NCBI menggunakan Primer-BLAST. Sebagai templat sekuen DNA yang digunakan dalam analisis primer diunduh dari situs www.ncbi.nlm.nih.gov dengan accession number: MK579314.1 yang merupakan sekuens DNA gen embB M. tuberculosis H37Rv. Kesimpulan yang diperoleh dari penelitian ini adalah, diperoleh pasangan primer forward gen embB dengan urutan sekuen 5’-ATTGCCCAGCTCCTCCTCAG-3’ (20 nukleotida) dan primer reverse dengan urutan sekuens 5’-CGCCGCAACATGATGAACAC-3’ (20 nukleotida). Primer yang dihasilkan telah memenuhi kriteria pasangan primer yang baik dilihat dari panjang primer, nilai Tm, %GC, stabilitas, jumlah hairpins, repeats, dimers, dan runs. Serta, hasil uji spesifitas yang menunjukan pasangan primer spesifik mengenali hanya bakteri M. tuberculosis.
Kata kunci: Desain primer, gen embB, in silico, M. tuberculosis, Resistensi Ethambutol
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