|Samira Mohammadi, Bahram Nasr Esfahani, Sharareh Moghim, Hossein Mirhendi, Fatemeh Riyahi Zaniani, Hajieh Ghasemian Safaei, Hossein Fazeli, Mahshid Salehi
Adv Biomed Res 2017, 6:133 (25 October 2017)
Background: Nontuberculous mycobacteria (NTM) are a group of opportunistic pathogens and these are widely dispersed in water and soil resources. Identification of mycobacteria isolates by conventional methods including biochemical tests, growth rates, colony pigmentation, and presence of acid-fast bacilli is widely used, but these methods are time-consuming, labor-intensive, and may sometimes remain inconclusive. Materials and Methods: The DNA was extracted from NTM cultures using CTAB, Chelex, Chelex + Nonidet P-40, FTA® Elute card, and boiling The quantity and quality of the DNA extracted via these methods were determined using UV-photometer at 260 and 280 nm, and polymerase chain reaction (PCR) amplification of the heat-shock protein 65 gene with serially diluted DNA samples. Results: The CTAB method showed more positive results at 1:10–1:100,000 at which the DNA amount was substantial. With the Chelex method of DNA extraction, PCR amplification was detected at 1:10 and 1:1000 dilutions. Conclusions: According to the electrophoresis results, the CTAB and Chelex DNA extraction methods were more successful in comparison with the others as regard producing suitable concentrations of DNA with the minimum use of PCR inhibitor.
|Sediqe Karimi, Hossein Mirhendi, Fatemh Riyahi Zaniani, Soroor Erfani Manesh, Mahshd Salehi, Bahram Nasr Esfahani
Adv Biomed Res 2017, 6:126 (16 October 2017)
Background: Molecular methods for the detection of drug-resistant tuberculosis (DR-TB) are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with DR-TB. The aim of this study was to evaluate and develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays for the detection of mutations within rpsL, and for the determination of streptomycin (STR) resistance in Mycobacterium tuberculosis. Materials and Methods: Clinical specimens were collected from individuals with suspected TB referred to the TB Center of Isfahan' from which 205 M. tuberclosis were isolated and identified by conventional phenotypic methods. The minimum inhibitory concentration of STR for all isolates was determined using the proportion method and 10 isolates were recognized as STR resistant M. tuberculosis. The effect of genetic alterations in the rpsL gene for these resistant isolates were investigated by PCR-RFLP method. Results: Three (30%) isolates showed point mutation at codon 43 by RLFP analysis. Conclusion: Our results suggest that RFLP analysis of the rpsL gene is useful for the rapid prediction of STR resistant strains of M. tuberculosis.