Authors:M. Naghavi, M. Ranjbar, A. Zali, M. Aghaei, M. Mardi, and S. Pirseyedi
Simple sequence repeat (SSR) DNA markers were used to characterize the genetic diversity in 70 accessions of
from Iran as well as to determine relationships among these accessions with 9 accessions of
) and 5
landraces. All twenty SSR primer pairs were polymorphic and identified a total number of 149 alleles corresponding to an average of 7.5 alleles per locus. The highest and lowest PIC values were obtained in subsp.
accessions, respectively. Data obtained were used to estimate genetic similarity using the Dice coefficient, and dendrogram was constructed using the UPGMA method. The dendrogram separated the 84 accessions into two main groups. All species grouped according to their genomes. A good level of genetic diversity was observed in the accessions of
, even in geographically close regions, which can be used in the broadening of the genetic base of bread wheat. In addition,
were clustered further away from
, confirming probably chromosomal rearrangements in the Dgenome of
during the processes of evolution.
Authors:M. Arjomandzadegan, P. Owlia, R. Ranjbar, A. Farazi, Masume Sofian, Maryam Sadrnia, Larisa Surkova, and L. Titov
Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.
Authors:M. Arjomandzadegan, P. Owlia, R. Ranjbar, A. Farazi, Masume Sofian, Maryam Sadrnia, E. Ghaznavi-Rad, Larisa Surkova, and L. Titov
Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or “quiet” mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.