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

<p>Abstract</p> <p>Background</p> <p>The oomycete <it>Aphanomyces astaci </it>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, <it>A. astaci </it>is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for <it>A. astaci</it>, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.</p> <p>Results</p> <p>Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the <it>A. astaci </it>strain Gb04. The primary amino acid sequence of these chitinase genes, termed <it>CHI2 </it>and <it>CHI3</it>, 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 <it>A. astaci </it>mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state <it>CHI2 </it>and <it>CHI3 </it>mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for <it>CHI2 </it>and <it>CHI3</it>, respectively, is in accordance with the multifunctionality of GH18 family members. To identify <it>A. astaci</it>-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes <it>A. frigidophilus</it>, <it>A. invadans</it>, <it>A. helicoides</it>, <it>A. laevis</it>, <it>A. repetans</it>, <it>Achlya racemosa</it>, <it>Leptolegnia caudata</it>, and <it>Saprolegnia parasitica</it>, as well as in the relevant fungi <it>Fusarium solani </it>and <it>Trichosporon cutaneum</it>. An <it>A. astaci</it>-specific primer pair targeting the novel genes <it>CHI2 </it>and <it>CHI3 </it>as well as <it>CHI1 </it>- a third GH18 family member - was multiplexed with primers targeting the <it>5.8S rRNA </it>used as an endogenous control. A species was typed unambiguously as <it>A. astaci </it>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.</p> <p>Conclusion</p> <p>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 (<it>CHI1</it>, <it>CHI2</it>, <it>CHI3</it>, 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.</p> <p>Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.</p