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Background  The oomycete  Aphanomyces astaci  is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently,  A. astaci  is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for  A. astaci , which achieves robustness through simultaneous amplification of multiple functionally constrained genes.    Results  Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the  A. astaci  strain Gb04. The primary amino acid sequence of these chitinase genes, termed  CHI2  and  CHI3 , is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The  A. astaci  mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state  CHI2  and  CHI3  mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for  CHI2  and  CHI3 , respectively, is in accordance with the multifunctionality of GH18 family members. To identify  A. astaci -specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes  A. frigidophilus ,  A. invadans ,  A. helicoides ,  A. laevis ,  A. repetans ,  Achlya racemosa ,  Leptolegnia caudata , and  Saprolegnia parasitica , as well as in the relevant fungi  Fusarium solani  and  Trichosporon cutaneum . An  A. astaci -specific primer pair targeting the novel genes  CHI2  and  CHI3  as well as  CHI1  - a third GH18 family member - was multiplexed with primers targeting the  5.8S rRNA  used as an endogenous control. A species was typed unambiguously as  A. astaci  if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.    Conclusion  The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes ( CHI1 ,  CHI2 ,  CHI3 , and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods.  Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci