Mycobacterium Tuberculosis Complex Strains Identification with Spoligotyping Method in Patients Attending to Masih Daneshvari Hospital

Background and Objective: Spoligotyping is a method based on ۳۶bp Direct Repeat (DR) chromosomal loci polymorphism which is connected to one or two ۳۵-۴۱ bp spacer sequences. There are ۹۴ different intra DR spacer sequences which are identified so far and only ۴۳ of them are used as usual. Mycobacterium tuberculosis complex strains can be identified based on lacking or having these sequences. Materials and Methods: Spoligotyping test was carried out on ۲۳۸ TB smear positive patients. Primary separation of mycobacterium strains was done through Petrof ۴% method and Lowenstein Jensen (LJ) media. Biochemical tests such as Niacin test/Catalase activity/Nitrate reduction were done in order to identify the strains. Drug sensitivity to INH (۰.۲Mg/ml)/ RIF (۴۰Mg/ml)/ STM (۱۰Mg/ml) and ETBl (۲Mg/ml) identified by proportional method and according to that, the strains were divided into three groups: sensitive, multi drug resistance (MDR) and non MDR. Then DNA was extracted by CTAB method from the positive colonies. Sequences were amplified by PCR and after denaturizing, hybridization with Streptavidine peroxidase enzyme was performed by Line reverse blot method. Radiography was done after adding the Luminoscense and membrane onto the X-ray films. Results: Serotypes were divided into ۹ groups (Beijing/ CAS۱/ Haarlem / U/ T۲/ T۱/ EAI۳/ EAI۲ and CAS۲). Most of the strains were from Haarlem (۲۷%) and CAS۱ (۲۵%) groups. Two strains were also identified in this method that belonged to Mycobacterium bovis. Conclusion: Spoligotyping method is an easy, rapid and sensitive test in order to identify Mycobacterium tuberculosis complex strains.

Keywords:

Keywords: Spoligotyping , Mycobacterium Tuberculosis , TB patients