Molecular Identification of Mycobacterium tuberculosis Complex by RD-Typing and PCR-RFLP Method

Document Type : Full Research Paper

Authors

1 Department of Microbiology, Qom Branch, Islamic Azad University, Qom, Iran.

2 Bovine tuberculosis reference laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extention Organization (AREEO), Karaj, IRAN.

Abstract

Tuberculosis is still considered as one of the most important zoonotic diseases and the cause of infectious death and human casualties. The main purpose of this study was to set up and diagnose Mycobacterium tuberculosis complex isolates in patients of FARS, Shiraz using the Regions of Difference (RD) typing and RFLP techniques. The genomes of 50 isolates were extracted by boiling method. To determine the genus of the isolates, PCR technique based on 16 s rRNA was used. IS6110 PCR and RD typing were used for the detection of Mycobacterium tuberculosis complex. Finally, the PCR-RFLP method was used as a comparing method for comparative evaluation of RD typing to identify mycobacteria. Amplification of 543 bp bands in 16s rRNA PCR showed that all studied isolates belonged to the genus Mycobacterium. In IS6110 gene PCR, it was shown that all isolates were Mycobacterium tuberculosis complex due to the amplification of 245 bp bands. In RD typing the identification was based on RD1, RD4, RD9 and RD12. The comparative method of PCR-RFLP on pseudo gene oxyR showed that all isolates were Mycobacterium tuberculosis. Using RD-typing, it was found that all isolates achieved from Shiraz were Mycobacterium tuberculosis. The absence of Mycobacterium bovis isolates represents good hygiene and consumption of pasteurized milk in this city. The results of this study also reflect the ability of RD typing technique in differentiation of mycobacteria.

Keywords

References

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Veterinary Research & Biological Products
Volume 30, Issue 3 - Serial Number 116
September 2017
Pages 16-25

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History

  • Receive Date: 17 August 2016
  • Revise Date: 14 November 2016
  • Accept Date: 21 November 2016

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