Hülya ŞİMŞEK, Serdar TUNCER, Gülnur TARHAN, İsmail CEYHAN

5166

Evaluation of the Efficacy of Five DNA Extraction Methods for the Detection of Mycobacterium tuberculosis DNA in Direct and Processed Sputum by an In-House PCR Method

Aim: DNA extraction is an important step from clinical samples for molecular diagnosis of tuberculosis by PCR. The aim of this study was to evaluate the efficacy of five DNA extraction methods [boiling, single step proteinase K, GuSCN lysis and izopropanol precipitation (Heliosis, METİS Company, TURKEY), DNA precipitation (Epicentre Technologies), and solid phase absorption (QIAamp DNA mini kit, QIAGEN, Valancia,CA)] in searching Mycobacterium tuberculosis DNA in smear positive sputum samples. Materials and Methods: A total of 50 sputum samples were extracted directly and after digested with 4% NaOH-NALC methods using 5 DNA extraction methods. All DNA extracts were studied by an in-house PCR method. Results: The rate of the positive detection for 5 extraction methods was 22% with boiling method, 38% with single step proteinase K, 38% with guanidium isothiocyanate lysis and isopropanol precipitation method (Heliosis, METIS ), 42% with DNA precipitation (Epicentre Technologies), and 58% with solid phase absorption (QIAamp). Conclusions: When the rate of positive detection is taken into consideration in smear positive patients, solid phase absorption method seems to be more proper to use routinely for DNA isolation from clinical samples.
Anahtar Kelimeler:

DNA extraction, Mycobacterium tuberculosis, PCR

Evaluation of the Efficacy of Five DNA Extraction Methods for the Detection of Mycobacterium tuberculosis DNA in Direct and Processed Sputum by an In-House PCR Method

Aim: DNA extraction is an important step from clinical samples for molecular diagnosis of tuberculosis by PCR. The aim of this study was to evaluate the efficacy of five DNA extraction methods [boiling, single step proteinase K, GuSCN lysis and izopropanol precipitation (Heliosis, METİS Company, TURKEY), DNA precipitation (Epicentre Technologies), and solid phase absorption (QIAamp DNA mini kit, QIAGEN, Valancia,CA)] in searching Mycobacterium tuberculosis DNA in smear positive sputum samples. Materials and Methods: A total of 50 sputum samples were extracted directly and after digested with 4% NaOH-NALC methods using 5 DNA extraction methods. All DNA extracts were studied by an in-house PCR method. Results: The rate of the positive detection for 5 extraction methods was 22% with boiling method, 38% with single step proteinase K, 38% with guanidium isothiocyanate lysis and isopropanol precipitation method (Heliosis, METIS ), 42% with DNA precipitation (Epicentre Technologies), and 58% with solid phase absorption (QIAamp). Conclusions: When the rate of positive detection is taken into consideration in smear positive patients, solid phase absorption method seems to be more proper to use routinely for DNA isolation from clinical samples.
Keywords:

DNA extraction, Mycobacterium tuberculosis, PCR,

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Turkish Journal of Medical Sciences-Cover
  • ISSN: 1300-0144
  • Yayın Aralığı: 6
  • Yayıncı: TÜBİTAK
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