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. 2015 Jul 27:4:152.
doi: 10.4103/2277-9175.161579. eCollection 2015.

Molecular typing of Iranian mycobacteria isolates by polymerase chain reaction-restriction fragment length polymorphism analysis of 360-bp rpoB gene

Shima Hadifar  1 , Sharareh Moghim  1 , Hossein Fazeli  1 , Hajieh GhasemianSafaei  1 , Seyed Asghar Havaei  1 , Fariba Farid  2 , Bahram Nasr Esfahani  1
Affiliations

Molecular typing of Iranian mycobacteria isolates by polymerase chain reaction-restriction fragment length polymorphism analysis of 360-bp rpoB gene

Shima Hadifar et al. Adv Biomed Res. .

Abstract

Background: Diagnosis and typing of Mycobacterium genus provides basic tools for investigating the epidemiology and pathogenesis of this group of bacteria. Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) is an accurate method providing diagnosis and typing of species of mycobacteria. The present study is conducted by the purpose of determining restriction fragment profiles of common types of mycobacteria by PRA method of rpoB gene in this geographical region.

Materials and methods: Totally 60 clinical and environmental isolates from February to October, 2013 were collected and subcultured and identified by phenotypic methods. A 360 bp fragment of the rpoB gene amplified by PCR and products were digested by MspI and HaeIII enzymes.

Results: In the present study, of all mycobacteria isolates identified by PRA method, 13 isolates (21.66%) were Mycobacterium tuberculosis, 34 isolates (56.66%) were rapidly growing Nontuberculosis Mycobacteria (NTM) that including 26 clinical isolates (43.33%) and 8 environmental isolates (13.33%), 11 isolates (18.33%) were clinical slowly growing NTM. among the clinical NTM isolates, Mycobacterium fortuitum Type I with the frequency of 57.77% was the most prevalent type isolates. Furthermore, an unrecorded of the PRA pattern of Mycobacterium conceptionense (HeaIII: 120/90/80, MspI: 120/105/80) was found. This study demonstrated that the PRA method was high discriminatory power for identification and typing of mycobacteria species and was able to identify 96.6% of all isolates.

Conclusion: Based on the result of this study, rpoB gene could be a potentially useful tool for identification and investigation of molecular epidemiology of mycobacterial species.

Keywords: Molecular typing; mycobacteria; polymerase chain reaction-restriction fragment length polymorphism analysis; rpoB gene.

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Conflict of interest statement

Conflict of Interest: None declared.

Figures

Figure 1
Figure 1
(a) Result of polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) studied mycobacteria lanes: M, size marker (50-bp); 1–7, Mycobacterium fortuitum Type I; 8, unknown 9, Mycobacterium kansasil Type L; 10, Mycobacterium smegmatis, 11, Mycobacterium avium, 12–13, Mycobacterium gordonae Type II. (b) Result of PRA studied mycobacteria lanes: M, size marker (50-bp); 1–6, Mycobacterium fortuitum Type I; 7, Mycobacterium intracelluare; 8, Mycobacterium conceptionese, 9, Mycobacterium kansasii Type 1; 10, Mycobacterium fortuitum Type I, 11–13, Mycobacterium gordonae Type I
See this image and copyright information in PMC

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