Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

ABSTRACT: Background: Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods: Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results: Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions: The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings.

• Autores: Arango Flórez, Eliana María; Agudelo García, Olga María; Maestre Buitrago, Amanda Elena; Yanow, Stephanie K; Martin, Kimberly A; Taylor, Brian J
• Tipo de recurso: Article of investigation
• Fecha de publicación: 2011
• Institución: Universidad de Antioquia
• Repositorio: Repositorio UdeA
• Idioma: eng
• OAI Identifier: oai:bibliotecadigital.udea.edu.co:10495/34773
• Acceso en línea: https://hdl.handle.net/10495/34773
• Palabra clave: Sangre - parasitología; Blood - parasitology; Técnicas de Laboratorio Clínico - métodos; Clinical Laboratory Techniques - methods; ADN Protozoario; DNA, Protozoan; Malaria; Parasitemia; Plasmodium; Reacción en Cadena en Tiempo Real de la Polimerasa - métodos; Real-Time Polymerase Chain Reaction - methods
• Rights: openAccess
• License: http://creativecommons.org/licenses/by/2.5/co/