Detection of INH Target Genes on DNA of Positive Acid Fast Stain Sputum Using PCR Technique

Tuberculosis, the world’s major diseases, is one of the emerging infectious diseases . The tuberculosis problem has become complicated and burdensome due to the emergence of drug resistan such as isoniazid (INH) resistant strains of Mycobacterium tuberculosis. The resistance can be detected by analyzing the genes which code the INH target genes such as kat-G and kas-A. Research to investigate the existence of the genes has being carried out on 25 positive acid fast stain sputum samples. The extraction of sputum DNA was done by means of three kind of methods including Boom’s method, proteinase-K, and boiling methods. PCR technique with two pairs of the oligonucleotide primers for kat-G and kas-A genes, subsequently continued with the agarose gel electrophoresis process, were used for detecting the existence of the genes. Results revealed that the existence of kat-G gene can be detected on 24 of the 25 sputum samples DNA which extracted by either Boom’s or proteinase-K methods, 1 sample were negative result, and by using boiling method, 3 samples showed the negative results. However, the DNA bands of 1 and 5 samples from 25 positive samples mentioned above, appeared to be thin bands by using Boom’s and proteinase- K, respectively. The existence of kas-A gene showed the positive results on 24 samples of the 25 sputum samples DNA and 1 sample was negative by means of Boom’s method, whereas with proteinase-K and boiling methods , the negative results appeared on 7 and 16 sputum samples, respectively. From the results mentioned above, it can be concluded that the best DNA extraction method is Boom’s method. However, the more simple method that is as same as Boom’s method, is necessary to be carried out.