A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis

Revista da Sociedade Brasileira de Medicina Tropical

Maria Almerice Lopes Da Silva¹  Zulma Medeiros¹  Cynthia Regina Pedrosa Soares¹  Elis Dionísio Da Silva¹  Demócrito Barros Miranda-filho¹  Fábio Lopes De Melo¹ 

关键词: Urine;    Extraction;    DNA;    Visceral leishmaniasis;   

DOI  :  10.1590/0037-8682-0233-2013

来源: SciELO

【 摘 要 】

Introduction Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.

【 授权许可】

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