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IJHSR. 2016; 6(1): 450-458


Rapid Detection of Mycobacterium Tuberculosis Complex with IS6110 Marker Based on Real Time PCR High Resolution Melting Analysis.

Girish Pai, Mansee Thakur, Harapriya Kar, D.S.Joshi.

Abstract
Mycobacterium tuberculosis (MTB) is a major agent of infection for human tuberculosis (TB) worldwide. Although T.B. treated by a combination of six-monthly course of antibiotics, the rise in prevalence of drug-resistance TB (MDR-TB, XDR-TB) makes the it global health problem. Molecular methods have a significant role to play in MTB and drug resistance TB. Recently, high-resolution melting (HRM) Real time PCR analysis was used for amplicon genotyping and mutation scanning. The present study aims to develop a diagnostic method for MTB detection based on HRM analysis that does not require labeled oligonucleotides. Polymerase Chain Reaction was performed for MTB detection with IS6110 as a target gene. Derivative melting curves of the IS 6110 specific target amplified duplexes were characteristic of the genotype of MTB and non tubercular clinical isolates. On fluorescence analysis, Light cycler 480 gene scanning software analyzes and distinguishes presence or absence of MTB depending on the shape of the curve. We collected 100 suspected clinical isolates, including 10 non tuberculosis cases. These samples were analyzed for acid-fast bacilli (AFB), culture & Real Time PCR. The sensitivity and specificity of detecting MTB by HRM Real Time PCR was 98.51% and 93.94% respectively. This technique requires the unlabeled primers specific to the target sequence and a DNA specific intercalating dye which can prove a promising method for detection of Mycobacterium tuberculosis.

Key words: Mycobacterium tuberculosis, HRM, RT PCR, IS6110.



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