Development of Sequence Based Molecular Diagnostic Test to Evaluate MDR and XDR in M. tuberculosis Patients from Western India

摘要

Globally, Tuberculosis is the most distress disease which remains as a major challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. The increasing knowledge in molecular biology techniques has improved our understanding of the molecular mechanisms of drug resistance for the major first and second-line anti- tubercular drugs. Thus, rapid and accurate diagnosis of Tuberculosis would improve patient care and limit its transmission. This study is aimed to identify mutations in drug resistance genes of first and second line drugs against Mycobacterium tuberculosis by using polymerase chain reaction (PCR) and DNA sequencing methods. This study involves sputum samples of 20 patients diagnosed to have pulmonary tuberculosis. The genomic DNA from clinical isolates was isolated using simple boiling method. PCR amplification and DNA sequencing were performed for katG, inhA, ndh, kasA, pncA, embB, rrs, rpsL, rpoB, gyrA, gyrB and thyA genes. Sequence analysis was done using Bioedit, a bioinformatics tool. The isolates of Mycobacterium tuberculosis from western part of India were analyzed by DNA sequencing revealed total 29 mutations in first line drugs. Out of them, 5(25%) in rpoB at codon 516 and 531, 6(30%) in katG at codon 315, 8(40%) in rpsL at codon 43 & 88, 3(15%) in embB at codon 306 and 7(35%) in pncA at codon 195.No mutations in inhA, ndh, kasA and rrs genes were observed in our study. In the second line drugs, 2(10%) at codon 90 & 100% mutations at codon 95 in gyrA gene, and no mutation in gyrB and thyA genes were observed.