Optimization of Annealing Temperature for Amplification of 507 bp fragment of rpoB Gene of Clinical Multidrug-Resistant Mycobacterium tuberculosis Isolate 86

Abstract

The gene of rpoB is the primary gene that is well known as a surrogate marker in MDR-TB (Multidrug-Resistant Tuberculosis) detection. The mutation of rpoB was studied around the world in its core region called RRDR (Rifampicin Resistance Determining Region). To learn the mutation in this fragment, PCR (Polymerase Chain Reaction) is the most common method used. This study was purposed to optimize the annealing temperature in amplifying the 507 bp fragment of rpob gene containing RRDR of isolate 86.DNA of MDR-TB isolate 86 was isolated by using Boom method for further amplified by PCR. The oligonucleotide primers used in this study were FrTB and RrTB. Eight different annealing temperature were used to optimize the amplification of rpoB gene : 53, 55, 57, 58, 59, 60, 61, and 63oC. Detection of PCR products was done with 1,5% agarose gel electrophoresis.Agarose gel electrophoresis of PCR products showed that 507 bp of rpoB gene could be produced at all annealing temperatures. A faint amplified product was observed at temperature 53 and 55oC. However, at temperature 58 and 60oC more intense bands were observed. In conclusion, the best annealing temperature was at 60oC in producing the 507 bp fragment.