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Currently, <jats:italic>A. astaci<\/jats:italic> is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for <jats:italic>A. astaci<\/jats:italic>, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the <jats:italic>A. astaci<\/jats:italic> strain Gb04. The primary amino acid sequence of these chitinase genes, termed <jats:italic>CHI2<\/jats:italic> and <jats:italic>CHI3<\/jats:italic>, 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 <jats:italic>A. astaci<\/jats:italic> mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state <jats:italic>CHI2<\/jats:italic> and <jats:italic>CHI3<\/jats:italic> mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for <jats:italic>CHI2<\/jats:italic> and <jats:italic>CHI3<\/jats:italic>, respectively, is in accordance with the multifunctionality of GH18 family members. To identify <jats:italic>A. astaci<\/jats:italic>-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes <jats:italic>A. frigidophilus<\/jats:italic>, <jats:italic>A. invadans<\/jats:italic>, <jats:italic>A. helicoides<\/jats:italic>, <jats:italic>A. laevis<\/jats:italic>, <jats:italic>A. repetans<\/jats:italic>, <jats:italic>Achlya racemosa<\/jats:italic>, <jats:italic>Leptolegnia caudata<\/jats:italic>, and <jats:italic>Saprolegnia parasitica<\/jats:italic>, as well as in the relevant fungi <jats:italic>Fusarium solani<\/jats:italic> and <jats:italic>Trichosporon cutaneum<\/jats:italic>. An <jats:italic>A. astaci<\/jats:italic>-specific primer pair targeting the novel genes <jats:italic>CHI2<\/jats:italic> and <jats:italic>CHI3<\/jats:italic> as well as <jats:italic>CHI1<\/jats:italic> - a third GH18 family member - was multiplexed with primers targeting the <jats:italic>5.8S rRNA<\/jats:italic> used as an endogenous control. A species was typed unambiguously as <jats:italic>A. astaci<\/jats:italic> 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.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats: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 (<jats:italic>CHI1<\/jats:italic>, <jats:italic>CHI2<\/jats:italic>, <jats:italic>CHI3<\/jats:italic>, 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.<\/jats:p>\n            <jats:p>Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1471-2180-9-184","type":"journal-article","created":{"date-parts":[[2009,9,1]],"date-time":"2009-09-01T06:14:25Z","timestamp":1251785665000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":27,"title":["Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci"],"prefix":"10.1186","volume":"9","author":[{"given":"Gerald","family":"Hochwimmer","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reinhard","family":"Tober","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ren\u00e8","family":"Bibars-Reiter","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elisabeth","family":"Licek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ralf","family":"Steinborn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2009,8,31]]},"reference":[{"issue":"1","key":"855_CR1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/j.1574-6968.2007.00786.x","volume":"274","author":"KH Lamour","year":"2007","unstructured":"Lamour KH, Win J, Kamoun S: Oomycete genomics: new insights and future directions. 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