Identification of Non-Tuberculosis Mycobacteria Isolates by Polymerase Chain Reaction-Restriction Enzyme Analysis of 360-Bp Fragment of Rpob Gene



Hadifar S¹ ; Moghim S² ; Gasemiansafaei H² ; Fazeli H² ; Farid F³ ; Moghoofei M¹ ; Sedighi M¹ ; Nasresfahani B² Show Affiliations
Source: Journal of Isfahan Medical School Published:2014

Abstract

Background: Diagnosis of mycobacterium genus provides a basis for investigating the epidemiology and pathogenesis of this group of bacteria. Regarding the prevalence of mycobacterial infection in Iran and because of the being neighborhood with countries among 22 high-burden countries, increasing attention to mycobacterial diseases and introducing molecular epidemiology of Mycobacteria seems to be necessary. Restriction Fragment Length Polymorphism Analysis of Polymerase Chain Reaction- Amplified Fragments (PCR-RFLP) is an inexpensive and accurate method providing diagnosis of mycobacterial species. The present study aimed to determine the common types of Mycobacteria in this geographical region by mentioned method. Methods: 34 clinical isolates were collected and cultured and identified by phenotypic methods. A 360-bp fragment of the rpoB gene was amplified by PCR and then, PCR products were digested by the two enzymes, MspI and HaeIII. Digested fragments were analyzed by using 4% metaphor agarose gel electrophoresis. Findings: Out of 34 species of nontuberculous mycobacteria (NTMs), M. fortuitum type I with the frequency of 82.35% was the most frequent type and M. gordonae type I and M. kansasii type I both with the frequency of 5.88% and M. gordonae type II and M. intracellular both with the frequency of 2.94% were the next. Conclusion: The PCR-RFLP analysis of rpoB gene used for identification of Mycobacteria provided valid results in this geographical area. In this study, M. kansasii type I (HeaIII: 90/205, MspI: 30/40/60/175) and M. avium (HeaIII: 270; MspI: 40/80/105) were identical to the patterns of some studies and different from others. This study demonstrated the high sensitivity (100%) of used PCRRFLP analysis method for identification of Mycobacteria.