Evaluation of DNA Extraction Methods for Detection of <i>Leishmania</i> by Polymerase Chain Reaction
- 1 Faculty of Health Sciences, Universidade Federal da Grande Dourados (UFGD), Dourados, Brazil
- 2 Faculty of Health Sciences, Universidade Federal da Grande Dourados (UFGD), Dourados, Brazil
- 3 Postgraduate Program of Health Sciences, Universidade Federal da Grande Dourados (UFGD), Dourados, Brazil
- 4 Postgraduate Program of Health Sciences, Universidade Federal da Grande Dourados (UFGD), Dourados, Brazil
- 5 Aggeu Magalhães Institute, Oswaldo Cruz Fundation (Fiocruz), Recife, Brazil
Abstract
There are several methods used to obtain DNA from cells; however, the quantity, integrity, and purity of DNA vary among the methods, which may interfere with the polymerase chain reaction (PCR) results. The objective was to determine the most efficient and cost-effective method that provides the best DNA yield and PCR results. Three methods of DNA isolation were compared: 20% sodium dodecyl sulfate (SDS), guanidine isothiocyanate-phenol-chloroform (GTPC), and DNA extraction using a commercial kit (GE Healthcare GenomicPrep Blood DNA Isolation Kit TM ). Human peripheral blood samples were inoculated with 10 4 promastigotes of Leishmania infantum . DNA was quantified and PCR was performed with 13A/13B primers. The results showed that a higher DNA yield was obtained using the GTPC technique (214.51 ng/μL), followed by SDS (26.16 ng/μL) and the commercial kit (10.99 ng/μL). We concluded that while all of the techniques were effective for obtaining DNA, the GTPC method provided the best yield and the brightest bands.
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