Study of carD gene sequence in clinical isolates of Mycobacterium tuberculosis

Sarmadian, Hossein and Nazari, Razieh and Zolfaghari, Mohammad and Pirayandeh, Mina and Sadrnia, Maryam and Arjomandzadegan, Mohammad and Titov, Leonid and Rajabi, Fariba and Ahmadi, Azam and Shojapoor, Mana (2014) Study of carD gene sequence in clinical isolates of Mycobacterium tuberculosis. Acta Microbiologica et Immunologica Hungarica, 61 (1). pp. 1-10. ISSN 1217-8950

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Abstract

Mycobacterium tuberculosis growth rate is closely coupled to rRNA transcription which is regulated through carD gene. The aim of this study was to determine the sequence of carD gene in drug susceptible and resistant clinical isolates of M. tuberculosis and designing of a PCR assay based on carD sequence for rapid detection of this bacterium.Specific primers for amplification of carD gene were carefully designed, so that whole sequence of gene could be amplified; therefore primers were positioned at the upstream (promoter of this gene and ispD gene) and downstream (in ispD gene). DNA from 41 clinical isolates of M. tuberculosis with different pattern of drug resistance was used in the study. PCR conditions and annealing temperature were designed by means of online programs. PCR products were sequenced by ABI system.PCR product of carD gene was a 524 bp fragment. This method could detect all resistant and susceptible strains of M. tuberculosis. The size of amplified fragment was similar in all investigated samples. Sequence analysis showed that there was similar sequence in all of our isolates therefore probably this gene is considered to be conservative. Translation of nucleotide mode to amino acids was showed that TRCF domain in N-terminal of protein CarD was found to be fully conservative.This is the first study on the sequence of carD gene in clinical isolates of M. tuberculosis. This conservative gene is recommended for use as a target for designing of suitable inhibitors as anti-tuberculosis drug because its importance for life of MTB. In the other hand, a PCR detection method based on detection of carD gene was recommended for rapid detection in routine test.

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