Abstract
Objective: Rapid confirmation of diagnosis of MTB and MDR-MTB in clinical samples by RT PCR and Melting Curve Analysis (MCA).
Introduction: TB is one of the leading causes of mortality in world. An extremely worrisome aspect of MTB is the recent rise in MDR- MTB cases in several countries. The culture based diagnostic procedures take weeks to detect TB and its drug resistant variants. In developing countries this delay could compromise efforts to interrupt TB transmission. There is, therefore, intense interest to develop rapid and precise molecular diagnostic methods.
Methods: DNA was extracted from sputum or body fluids of about 100 patients suspected of TB. MTB diagnosis was confirmed by RT PCR amplification and subsequent melting curve analysis, using IS6110 specific primers on Lightcycler480 (Roche Inc). For MDR MTB diagnosis hydrolysis probes were used to genotype mutations in rpoB and katG genes, associated with resistance to rifampin (RIF) and Isoniazide (INH) respectively.The data obtained from culture and molecular methods was compared.
Results: MTB diagnosis was confirmed by RTPCR with 100% sensitivity on sybrgreen format. For detection of MDR, complete agreement between diagnoses from RT-PCR and Culture was observed in 38 cases out of 40 cases. 2 false positive for RIF resistance were was observed. No false positive or false negative cases were observed for resistance to INH in this study.
Conclusions: RT PCR based assay with MCA has considerable promise for confirmation of diagnosis of MTB and MDR MTB. Development of more specific probes can further improve its diagnostic potential.
- © 2011 ERS
