DETEKSI MUTASI KODON 510 dan 511 DAERAH RRDR GEN rpoB PADA ISOLAT KLINIK Mycobacterium tuberculosis MULTIDRUG RESISTANT DI BALI DENGAN PCR-RESTRICTION FRAGMENT LENGHT POLYMORPHISM
Abstract
ABSTRAK: Tujuan penelitian ini adalah untuk melakukan deteksi mutasi daerah RRDR gen rpoB Mycobacterium tuberculosis khususnya pada kodon 510 dan 511 dari isolat klinis Multidrug Resistant Tuberculosis (MDR-TB) di Bali dengan metode Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP). Isolat M. tuberculosis H37Rv digunakan sebagai kontrol bakteri yang tidak mengalami mutasi dan empat isolat klinis MDR-TB digunakan sebagai sampel pada penelitian ini. Proses PCR-RFLP meliputi dua tahap, yaitu amplifikasi (PCR) dan digesti. Produk PCR hasil amplifikasi didigesti dengan enzim PvuII (New England Biolabs) melalui proses inkubasi pada suhu 37oC selama 3 jam diikuti dengan inaktivasi ice shock pada suhu -20oC selama 5 menit. Hasil penelitian ini menunjukan bahwa enzim restriksi PvuII dapat mendeteksi mutasi kodon 510 dan 511 daerah RRDR gen rpoB M. tuberculosis dengan teknik PCR-RFLP. Pada isolat 134 diketahui terdapat mutasi pada kodon 510 dan/atau 511 sedangkan pada isolat P10, P11, dan P16 tidak ditemukan adanya mutasi pada kodon 510 dan 511. Berdasarkan hasil penelitian sebelumnya, diketahui pula bahwa mutasi yang terjadi pada isolat 134 adalah mutasi kodon 510 (CAG?CTG).
ABSTRACT: The objective of this study is to detect mutation in the region of RRDR of rpoB gene Mycobacterium tuberculosis particularly at codon 510 and 511 from MDR-TB clinical isolates in Bali using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) method. Isolate of M. tuberculosis H37Rv was used as control of non-mutated bacteria, and four MDR-TB clinical isolates were used for sample in this study. PCR-RFLP was conducted in two steps which were amplification (PCR) and digestion. PCR products were digested using PvuII restriction enzyme (New England Biolabs) through incubation at 37oC for 3 hours followed by ice shock inactivation at -20oC for 5 minutes. The result of this study showed that PvuII restriction enzyme could detect mutation of codon 510 and 511 in the region of RRDR of rpoB gene M. tuberculosis using PCR-RFLP. In isolate 134, mutation at codon 510 and/or 511 was found while there were no mutation of codon 510 and 511 detected in isolate P10, P11, and P16. Based on previous research, it was found that the mutation occurred in isolate 134 was at codon 510 (CAG?CTG).
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