Rapid detection of mutations in codon 43 of rpsL gene by RFLP method with BsajI and MbooII restriction enzymes associated with resistance to Streptomycin in clinical isolates of Mycobacterium tuberculosis

Abstract

Rapid detection of mutations in codon 43 of rpsL gene by RFLP method with BsajI and MbooII restriction enzymes associated with resistance to Streptomycin in clinical isolates of Mycobacterium tuberculosis Author(s): Mohammad Arjomandzadegan1, Somaeah Geravand2, Azam Ahmadi *, Maryam Sadrnia4 , Manezheh Kahbazi5 1- Department of Microbiology and Immunology, Infectious Disease Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran 2- Department of Microbiology, Islamic Azad University, Tehran (Markazi), Iran 3- Department of molecular genetics, Tarbiat modares University, Infectious Disease Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran 4- Department of Biology, Payame Noor University, Tehran, Iran 5- Department of Pediatrics, Infectious Disease Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran Study Type: Research | Subject: General | Received: 2015/12/22 - Accepted: 2015/12/22 - Published: 2015/12/22 Abstract Background: Streptomycin is one of the most efficient treatments of tuberculosis that increasingly reported its drug resistance. The most common mutations associated with drug resistance to streptomycin of Mycobacterium Tuberculosis, causes tuberculosis, and occurs at codons 43 and 88 of rpsL gene. The purpose of this study is study of alterations in rpsL gene with two restriction enzymes BsajI and MbooII related to drug resistant to streptomycin in clinical isolates of mycobacterium tuberculosis. Materials and Methods: This study was performed using 71 clinical isolates of Mycobacterium tuberculosis. Molecular PCR-RFLP methods were designed with two enzymes BsajI and MbooII for mutation analysis at codon 43 of rpsL, in resistant and susceptible strains, respectively. Finally, the results were compared with sequencing and phenotype strains. Results: 25 subjects were studied with enzyme BsajI. This enzyme is capable of detection 64 percent of resistant and all sensitive strains. 46 strains were examined by enzyme MboII. This enzyme detected almost 91 percent of sensitive strains. MboII is selected for detection of sensitive strains (unlike BsajI). Results of sequencing rpsL genes in investigated strains, showed fully consistent with the results of PCR-RFLP and proved mutation in codon of 43 of thin genes in studied strains. Conclusion: The results showed that the PCR-RFLP with designed restriction enzymes can be used as a rapid, simple assay, and has high sensitivity for differentiation of Mycobacterium tuberculosis strains that are resistance to Streptomycin. Keywords: Drug resistance, Streptomycin, PCR-RFLP, BsajI,