title: A comparative study of molecular diagnostic methods designed to detect the crayfish plague pathogen, Aphanomyces astaci.
abstract: Crayfish plague is the most important disease of freshwater crayfish with a significant impact on European species. We compared the analytical test sensitivity and specificity of three published PCR assays for the detection of Aphanomyces astaci, the causative agent of crayfish plague: a conventional PCR assay targeting the ITS region and two TaqMan(®) real time assays, targeting either the ITS region or the chitinase gene. We also tested a variation of the conventional assay, by changing one of the primers. Test specificity was assessed using DNA from a range of A. astaci strains and an array of closely related Oomycetes, host tissue and DNA from other organisms that may be present in a diagnostic sample. Sensitivity was assessed using genomic A. astaci DNA from mycelium and zoospores. All assays were found to be of good to excellent sensitivity with levels of detection ranging from 1 (real time assay targeting the ITS region), over 10 (conventional PCR) to 100 zoospores (real time assay targeting the chitinase gene). All three published assays were also specific for A. astaci and did not cross-react with any other test organisms included in this study. The tested variation of the conventional PCR assay with a changed forward primer led to amplification of some non A. astaci DNA. Advantages and disadvantages, including suitable application are discussed for each assay.
authors.affiliation: Centre for Environment, Fisheries and Aquaculture Science, Barrack Road, Weymouth, Dorset, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Tuffs
authors.forename: Birgit
authors.initials: B
authors.lastname: Oidtmann
mesh_terms: D000818:Animals; D044744:Aphanomyces / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D017756:Aquaculture / Q000379:methods*; D003400:Astacoidea / Q000469:parasitology*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity