Identification of non-tuberculous mycobacteria isolated from respiratory specimens by PR-RFLPC method

Background: Rapid and accurate identification of mycobacteria is important for the treatment of the disease. Conventional methods used in identification have long been replaced by molecular methods with their controversial results and limitations in identifying over 170 species. In this study, it was aimed to identify 34 NTM isolates and 14 reference strains isolated from respiratory tract samples (sputum) sent from Adıyaman University Training and Research Hospital and from Atatürk Thoracic Diseases and Thoracic Surgery Training and Research Hospital (Doc. İsmail Ceyhan) according to the voluntary procedure at specify level by PCR-RFLP method. Material and methods: In our study, Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism (PCR-RFLP) method was used. It was ensured that the isolates coming to our laboratory were cultivated on LJ media. DNA isolation was performed by boiling the samples from the cultivated cultures and then amplified by PCR using primers specific to the hsp65 gene region. After amplification, PCR products were kept overnight with BstEII and HaEIII enzymes, and DNA fragments were evaluated by comparison with reference algorithms. Results: Species distribution of NTMs out of 34 isolates used in our study; 23.52% (8/34) M. intracellulare, 17.64% (6/34) M. gordonae, 17.64% (6/34) M. abscessus, 17.64% (6/34) M. simiae was determined as 5.88% (2/34) M. lentiflavum, 2.94% (1/34) M. kansasii and 2.94% (1/34) M. scrofulaceum. 4 of the samples could not be evaluated. Conclusion: The most common mycobacterial isolates were M. intracellulare, M. abscessus and M. simiae. According to this study, PCR-RFLP method is a useful method that provides faster and more precise diagnosis in comparison to conventional methods in the identification of mycobacteria at the species level. However, the steps of evaluating DNA fragments formed after enzyme cutting and comparing them with reference algorithms were found to be critical. It was concluded that standardization, especially in imaging processes, is very important in determining the correct type. Keywords: Non-tuberculosis Mycobacteria, PCR-RFLP, hsp65 gene