C: Quantitative detection of A. astaci by TaqMan qPCR. The standard curve of the assay demonstrates quantification down to 25 copies. The qPCR/MCA assay was tested for specifiCity against the oomycetes A. frigidophilus, A. invadans, A. laevis, A. helicoides, A. irregularis, and Leptolegnia caudata. Only the endogenous control was recorded, but not the A. astaci-specific chitinase peak. qPCR/MCA-based detection of A. astaci was used to elucidate several spontanous crayfish mortalities in Austrian waterbodies. In detail, A. astaci was identified as causative agent of acute crayfish-plague outbreaks among noble crayfish inhabiting a small unnamed pond-system (Hartberg, district Hartberg,

province Styria), in the noble crayfish-pond Bäckerteich (Velden am Wörthersee, Selleck Belnacasan district Villach-Land, province Carinthia), in the brook Hahntrattenbach near St. Andrä selleck screening library (district Wolfsberg, province Carinthia) known for its large stone crayfish population and in a noble crayfish population of the lake Gleinkersee (Roßleithen, district Kirchdorf an der Krems, province Upper Austria). A. astaci was also detected by MCA in necrobiopsy pools each derived from up to five euthanised signal crayfish specimens collected at the streams Ganaubach, Zöbernbach, Strem, Tauchenbach and Güns (province Burgenland). Clinical samples tested positive by MCA were subjected to pathogen isolation. In case of isolation

failure the qPCR/MCA amplicon was sequenced. TaqMan qPCR For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe-based qPCR assay was developed. TaqMan qPCR uses the same primers as qPCR/MCA except the additional nucleotide at the very 5′ end of the reverse primer compared to qPCR/MCA. Using amplicon standards with

known copy numbers spiked into genomic crayfish DNA, a quantitative detection limit of 25 target sequences was determined (Figure 5c). No amplication, i.e. C T > 50, was obtained for A. frigidophilus, A. invadans, A. leaevis and A. irregularis, In the case of the oomycete species A. helicoides and Leptolegnia caudata a cross-amplification signal corresponding to 28 and 44 copies was detected, respectively. Discussion Qualitative detection of two or multiple target sequences by MCA has been SSR128129E reported before. Single-tube SNP genotyping [41], sex determination [42], identification of methylation in promoter sequences [43] or the simultaneous detection of multiple pathogens [44, 45] are exemplarily mentioned. In this work we have used MCA of multiplex qPCR [46] for rapid species identification of the crayfish-plague pathogen A. astaci in a closed-tube format. The diagnostic assay for qualitative detection is highly discriminative, robust, inexpensive, and reliable. High Necrostatin-1 research buy discrimination was aimed at since new Aphanomyces ITS sequences, probably representing new Aphanomyces spp. and including sequences closely related to A. astaci were reported [47, 48].