Amplification of 0.7 Kb Fragment katG Gene from Clinical Multi-Drug Resistant Tuberculosis (MDR-TB) Isolate in Bali

The last decade has seen a particular increase in the occurrence of drugresistant (DR-TB) and multi-DR strains, such as Isoniazid (INH) resistant strains of M. tuberculosis. INH resistance is more frequently associated with mutations in the katG gene. Detection of katG gene mutations can be performed by PCR technique, followed by sequences. The aim of this study is to amplify katG gene region (0,7 Kb) from clinical isolate of MDR-TB in Bali. DNA isolation for PCR was done by Boom method and katG gene amplification was performed under the following conditions: predenaturation at 95C for 15 min; fourty cycles of denaturation at 94C for 1 min, annealing at 56C for 1 min, extension at 72C for 2 min; final extension at 72C for 10 min. The amplicons were detected by 1,5% agarose gel electrophoresis and showed a specific band size at 0.7 kb. This suggests that the fragment of katG gene has been successfully amplified in these area.


INTRODUCTION
In some studies, there are several options oligonucleotide primers katG gene region, a case of regional 209 bp, 351 bp and 580 bp 4,5,9 .In this study, we designed new primers with a longer area of amplification, that is 724 bp.
The frequency and type of mutations in the katG gene specific because of differences in geography 10,11

Agarose Gel Electrophoresis
The amplicons were detected by 1,5% agarose gel electrophoresis with TBE 1X and visualized by staining with 2,8 µL ethidium bromide.Running of electrophoresis was done at 65 V for 45 min.
Visualization was carried out by UV transilluminator (Gel Doc®).

RESULTS
The study begins with DNA    On the initial optimization condition, band is not formed.It can be caused by template concentrations is too low, the annealing temperature is too high, extension time is too short and lack of cycle 17 .
Template concentrations that are too low cause the primer can not find the target easily in a annealing process 18 .In this study, the Tuberculosis, caused by M. tuberculosis is one of the most important re-emerging infectious diseases 1 .Prevalence of tuberculosis in Indonesia (1990-2010) reached 289 cases per 100,000 population 2 .Particularly, in Bali, prevalence of TB cases occurred by 64 of 100,000 population based on a survey in 2004 3 .The last decade has seen a particular increase in the occurrence of drug-resistant (DR-TB) and multi-DR strains, such as Isoniazid (INH) resistant strains of M. tuberculosis 4 .Isoniazid (INH) is a prodrug which is converted into a biological active form by M. tuberculosis catalaseperoxidase, enzyme katG 5 .INH resistance in M. tuberculosis is caused by gene mutation that involves several genes, there are katG, inhA, oxyR-ahpC and kasA 6 .INH resistance is more frequently associated with mutations in the katG gene 7 .Mutation at codon 315 of katG gene is most prevalent in isoniazid resistant M. tuberculosis 8 .Detection of katG gene mutations can be performed by PCR technique, followed by sequencing.
primer (KG24F): 5' GAA GTA CGG CAA GAA GCT CTC 3' and reverse primer (KG60R): 5' CGT GAT CCG CTC ATA GAT CG 3'.The reaction of PCR was performed under the following conditions: pre denaturation at 95 o C for 15 min; fourty cycles of denaturation at 94 o C for 1 min, annealing at 56 o C for 1 min, extension at 72 o C for 2 min; final extension at 72 o C for 10 min.
isolation and continued with quality test of DNA to ensure DNA isolation outcome.Quality test of DNA done by PCR amplification using standard primer for M. tuberculosis.Result showed the band formed (Figure 1.) and means that positive results containing DNA.

Figure 1 .
Figure 1.PCR products using standard primers M. tuberculosis: M. marker (Invitrogen®) 100 bp DNA ladder, 1. fragment DNA from amplification of katG gene of clinical isolate M. tuberculosis

Figure 3 .
Figure 3. PCR product with annealing temperature at 56 o C: 1. is a fragment DNA (724 bp), M. marker (Invitrogen®) 100 bp DNA ladder concentration of DNA templates used by diluting 25 times in a initial optimization, so that need to increased template concentrations in a futher optimization by using template DNA concentration without dilution.The low quantity DNA may occur in this study is also the reason why the concentration of DNA template need to be improved.Annealing temperature is too high can cause the primer does not bind to the template 19 .Therefore, , it is needed to increase the extension time with the addition 1 min 17 .Therefore an extension time for further optimization increased from 1 min to 2 min.Lack of cycle amplification, especially on a low quantity of DNA will result in low DNA amplicons.So, it can not be detected by electrophoresis on agarose gel.Under these conditions, the addition of cycle needs to be done 3-5 cycles17 .So that, in a further optimization, the addition of the cycle was 35 cycles to 40 cycles.Based on the above, be reoptimized in order to obtain the band at 0.7 kb by increase sample concentration without diluted sample, decrease variation of annealing temperature be 48 o C up to 58 o C, increase extension time be 2 min and increase cycles of PCR be 40 cycles.Results from the further optimization conditions, produces a thin band with dimers at 48 o C, 50 o C and 56 o C. Whereas at 52 o C occurred misspriming and at 54 o C and 58 o C did not produce a band at 724 bp fragment.Annealing temperature at 48 o C and 50 o C produced specific band (724 bp) with thicker dimers than 56 o C. Thus, selected annealing temperature at 56 o C. The last stage with these conditions, amplification performed at 56 o C to make sure the band formed.Results showed that the thin band formed at 0.7 kb fragment (724 bp).CONCLUSION KatG gene region at 0.7 Kb fragment has been successfully amplified under the following conditions: pre denaturation at 95 o C for 15 min; fourty cycles of denaturation at 94 o C for 1 min, annealing at 56 o C for 1 min, extension at 72 o C for 2 min; final extension at 72 o C for 10 min.PCR amplification results showed thin band formed.Therefore, further research needs to be done to get ready for a thick band that was sequenced and help to detect katG gene mutations in region 0.7 kb.ACKNOWLEDGMENTS The author would like to thank Laboratory of Microbiology RSUP Sanglah to obtain clinical isolate of MDR-TB in Bali and Laboratory of Molecular Biology