title: Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.
abstract: Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.
authors.affiliation: Science and Conservation Division, Department of Parks and Wildlife, Perth, Australia; School Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia. Electronic address: judy.dunlop@dpaw.wa.gov.au.
authors.forename: J
authors.initials: J
authors.lastname: Dunlop
authors.affiliation: School Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia.
authors.forename: C K
authors.initials: CK
authors.lastname: Thompson
authors.affiliation: School Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia.
authors.forename: S S
authors.initials: SS
authors.lastname: Godfrey
authors.affiliation: School Veterinary and Biomedical Sciences, Murdoch University, Perth, Australia.
authors.forename: R C A
authors.initials: RC
authors.lastname: Thompson
mesh_terms: D000818:Animals; D003365:Costs and Cost Analysis; D016054:DNA, Protozoan / Q000097:blood* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards; D048328:Potoroidae; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013997:Time Factors; D014345:Trypanosoma / Q000235:genetics / Q000302:isolation & purification*; D014352:Trypanosomiasis / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan; D011933:Reagent Kits, Diagnostic
keywords: Blood; Disease; Infection; PCR; Parasite; Trypanosoma copemani