Microbiology
Faria Hasanzadeh Haghighi; Ehsan Aryan; Mohammad Derakhshan; Aida Gholoobi; Zahra Meshkat
Volume 13, Issue 4 , October 2018, , Pages 403-407
Abstract
Background & objective: Tuberculosis (TB) remains a major cause of death around the world. Bacillus Calmette Guérin (BCG) is the only vaccine used in TB prevention that has a protective effect in children, but its effectiveness declines in adults. Design and development of new vaccines ...
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Background & objective: Tuberculosis (TB) remains a major cause of death around the world. Bacillus Calmette Guérin (BCG) is the only vaccine used in TB prevention that has a protective effect in children, but its effectiveness declines in adults. Design and development of new vaccines is the most effective way against TB. The aim of this study was to design and construct a DNA vaccine encoding mtb32C and mpt51 fusion genes of Mycobacterium tuberculosis. Methods: First, mpt51 fragment was amplified by PCR method. The pcDNA3.1+/mtb32C plasmid was transformed into E. coli JM109 and then extracted. The mpt51 gene and pcDNA3.1+/mtb32C plasmid were both digested with EcoRI and BamHI restriction enzymes followed by ligation of mpt51 fragment into the digested vector. The recombinant plasmid containing mtb32C and mpt51 was subsequently transformed into competent E. coli TOP10 strain. The clones were confirmed by colony-PCR, restriction enzyme digestion and sequencing. Results: Using agarose gel electrophoresis, a 926 bp fragment corresponded to mpt51 was observed. Digestion of the vector pcDNa3.1+/mtb32C and mpt51 gene was confirmed by electrophoresis. Then, the pcDNA3.1+/mtb32C plasmid was extracted. Sequencing results confirmed the accuracy of the desired plasmid. Conclusion: In this study, we constructed a cloning vector encoding Mtb32C/Mpt51 gene of Mycobacterium tuberculosis. The eukaryotic expression of this vector can be confirmed in future studies. It can be considered as a DNA vaccine in animal models later. Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis.
Amir Tajbakhsh; Faezeh Ghasemi; Seyedeh Zohre Mirbagheri; Mastoureh Momen Heravi; Mehdi Rezaee; Zahra Meshkat
Volume 13, Issue 4 , October 2018, , Pages 429-437
Abstract
Background and Objectives: The incidence of rifampin-resistant strains of Mycobacterium tuberculosis has attracted more attention than the tuberculosis infection due to laborious treatment and control. Recognizing the Mycobacterium tuberculosis genotypes involving in drug resistance via multiplex PCR, ...
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Background and Objectives: The incidence of rifampin-resistant strains of Mycobacterium tuberculosis has attracted more attention than the tuberculosis infection due to laborious treatment and control. Recognizing the Mycobacterium tuberculosis genotypes involving in drug resistance via multiplex PCR, a simple and rapid genotyping method, is an emergency for better treatment and control of tuberculosis. This study was designed to specify the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from patients by multiplex allele-specific Polymerase Chain Reaction assay (MAS-PCR).Methods: In this study, 88 Mycobacterium tuberculosis positive samples were included from Qaem Hospital, Mashhad. MAS-PCR was used to detect the rifampin resistance associated mutations in rpoB gene. Results: Mutations in three codons of rpoB gene causing rifampin resistance were detected in 51 isolates (58.96%). The detected mutations in codons 531, 526, and 516 were 55.68%, 38.63%, and 13.63%, respectively. The simultaneous mutations were detected in 11 isolates (12.50%) in codons 531, 526 and 516, in 21 isolates (23.86%) in codons 531 and 526, and in one isolate (1.13%) in codons 526 and 516. Conclusion: According to the results of this study, the frequency of rifampin-resistant strains of Mycobacterium tuberculosis isolated from Khorasan province patients (North-East of Iran) was high. The developed MAS-PCR assay can be used for rapid detection in clinical diagnostic laboratories in areas with high prevalence of multidrug-resistant Mycobacterium tuberculosis strains. In this respect, MAS-PCR is simple, rapid, and highly sensitive method for drug susceptibility tests for detecting multidrug-resistant Mycobacterium tuberculosis.
Microbiology
Samira Rashidian; Roghayeh Teimourpour; Zahra Meshkat
Volume 11, Issue 2 , April 2016, , Pages 112-119
Abstract
Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid ...
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Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid and evaluation of its expression in eukaryotic cells. Methods: Firstly, tb10.4 fragment was amplified by PCR and the PCR product was digested with restriction enzymes. Next, it was cloned into pcDNA3.1+ plasmid. Following that, pcDNA3.1+/tb10.4 recombinant plasmid was transfected into eukaryotic cells. Results: 5700 bp band for pcDNA3.1+/tb10.4 recombinant plasmid and 297 bp fragment for tb10.4 were observed. Cloning and transfection were successful and designed recombinant vector was confirmed by sequencing. Conclusion: Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis. In the current study, the aim was cloning of tb10.4 gene in pcDNA3.1+ plasmid and transfection into eukaryotic cells.
