Differentiation of Mycobacterium tuberculosis clinical strains using restrikction fragment length polymorphism technique based on hsp65 gene

Polymerase chain reaction (PCR) coupled with Restriction Fragment Length Polymorphism analysis (RFLP) has been used for genotyping of Mycobacteria in recent years. While detailed differentiation of non tuberculosis Mycobacteria has been described by this technique, even in some species ability of this technique for molecular epidemiology has been proven, only a small number of Mycobacterium tuberculosis (MTB) strains have been studied. The present study was conducted to apply this method to an expanded population of MTB isolates. A total of 150 clinical isolates were collected from patients referred to TB reference unit, PHLS, Ahvaz, Iran. Acid fast staining was performed for the isolates and they were identified as MTB by using conventional culture and biochemical tests. The PCR-RFLP method uses a simple DNA extraction followed by a PCR step based on amplification of a 439 bp fragment of hsp65 gene involving genus specific primers. The restriction enzyme analysis was then performed by digestion of products with HaeIII and BstEII enzymes. Our results showed that 145 clinical isolates (96.6%) showed the identical restriction patterns similar to M. tuberculosis reference strains equal to 165/140/72 bp fragments for HaeIII and 250/120/82 bp fragments for BstEII digests. Three different patterns were observed for five clinical strains in HaeIII digest as 165/145 bp (three isolates), 180/100/80 bp (one isolate) and 194/72 bp (one isolate), while their BstEII digestion patterns showed no variation and were similar to other isolates. Our results showed that in clinical strains of mycobacterium tuberculosis, few polymorphisms in hsp65 fragment might be present