Objective: To create and evaluate the PCR restriction fragment length polymorphism (PCR-RFLP) based on hsp65 gene as a method for rapid identification of Mycobacteria to the species level.
Methods: hsp65 gene was amplified from the DNA of mycobacterial reference strains and the PCR products were subjected to digestion by two restriction endonucleases Hae III and Bstp I, then loaded onto a 4% MetaPhor agorose. The size of the restricted fragments of each species (strains) was determined according to the position of the fragments on the gel, by which the differential DNA fingerprint was confirmed.
Results: A total of 40 Mycobacterium species (strains) was analyzed, in which six reference strains of Mycobacterium tuberculosis Complex had two different electrophoresis patterns, and thirty-four reference species of non-tuberculosis Mycobacteria had unique pattern.
Conclusion: PCR-RFLP Based upon hsp65 gene can be used for identification of Mycobacterium species, and the method is more rapid and simple and easy-to-use for mycobacterial species identification.