Evaluation of DNA Extraction Methods for Detection of <i>Leishmania</i>by Polymerase Chain Reaction

• 作者: Carla Geórgia Rodrigues Guimarães Souto Herintha Coeto Neitzke-Abreu Letícia Surian Batalini Manoel Sebastião da Costa Lima-Junior Silvana de Oliveira Castro
• 来源期刊: 美国分子生物学期刊(英文)
• 学科: 医学
• 关键词: Leishmania DNA PURIFICATION PCR DIAGNOSIS
• 年，卷（期）: 2020,（4）
• 页码范围: 265-272
• 页数: 8页
• 分类号: R73

摘要

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 10⁴ promastigotes of <em>Leishmania infantum</em>. 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.