Use of 1.8kb Gene Amplification Restriction Analysis (PCR-RFLP) in Species Identification of M. tuberculosis.

Abstract: Conventional identification of a clinical isolate of Mycobacteria primarily based on culture characteristics and biochemical tests needs several weeks and may remain inconclusive. This study was undertaken to evaluate a new rapid method to identify the Mycobacterial isolates at species level by gene amplification restriction analysis using primers encoding 16S-23S rRNA internal transcribed spacer (ITS) region and flanking parts of the 16S as well as 23S rRNA gene. Restriction was carried out with restriction enzyme Hha I. This assay was applied on 5 reference strains and 50 clinical isolates of mycobacterial and 5 environmental isolates to validate the technique. Distinct gene amplification restriction analysis patterns were obtained by restriction of amplicons with Hha I restriction enzyme which could differentiate various mycobacterial species. The procedure dose not involve hybridization steps for the use of radioactivity and can be completed within 1 working day. Key words: Gene amplification restriction analysis, 16-23S rRNA- spacer region, Restriction enzyme