title: 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.
authors.affiliation: Department of Oncology, University of Alberta, Edmonton, Canada.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Taylor
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Martin
authors.forename: Eliana
authors.initials: E
authors.lastname: Arango
authors.forename: Olga M
authors.initials: OM
authors.lastname: Agudelo
authors.forename: Amanda
authors.initials: A
authors.lastname: Maestre
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Yanow
mesh_terms: D001769:Blood / Q000469:parasitology*; D019411:Clinical Laboratory Techniques / Q000379:methods; D003105:Colombia; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008288:Malaria / Q000175:diagnosis*; D018512:Parasitemia / Q000175:diagnosis*; D010274:Parasitology / Q000379:methods*; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*
chemical_list: D016054:DNA, Protozoan