Open Access Highly Accessed Research article

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

Gerald Hochwimmer1, Reinhard Tober2, Renè Bibars-Reiter13, Elisabeth Licek4 and Ralf Steinborn2*

Author Affiliations

1 Institute of Bacteriology, Mycology and Hygiene, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria

2 Vetomics Core Facility, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria

3 Department of Biochemistry, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria

4 Clinic for Avian, Reptile and Fish Medicine, University of Veterinary Medicine, Veterinaerplatz 1, 1210 Vienna, Austria

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BMC Microbiology 2009, 9:184  doi:10.1186/1471-2180-9-184

Published: 31 August 2009

Additional files

Additional file 1:

Species identification of Austrian A. astaci strains Gb04, Z12, and GKS07 based on phylogenetic analysis and constitutive chitinase activity in substrate-free medium. ITS sequence and chitinase expression in chitin-free medium are criteria to classify a strain as A. astaci

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Additional file 2:

Sequences of 3' untranslated regions (UTRs) of CHI2 and CHI3 mRNAs. Alignment shows differences between 3' UTRs of CHI2 and CHI3 mRNAs

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Additional file 3:

Amino-acid substitutions in the GH18 catalytic site of oomycete species. Table lists amino-acid substitutions in the GH18 catalytic site of oomycete species

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Additional file 4:

O-linked glycosylation and phosphorylation predicted for Chi2 and Chi3. Predicted O-linked glycosylations and phosporylations at serine and threonine residues for Chi2 and Chi3 are listed in a table

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Additional file 5:

Alignment of primer target sites for the 5.8S rRNA gene used as endogenous control in qPCR/MCA. Primers target conserved sites in the 5.8S rRNA gene of various oomycete species

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Additional file 6:

A conventional PCR assay for detection of A. astaci that may fail to discriminate between closely related species. Alignment of primer sites for a conventional PCR assay reported for detection of A. astaci

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Additional file 7:

Design of a homologous IPC for use in the qPCR/MCA or qPCR assays. The IPC monitored by a characteristic melting temperature or by an alternatively labeled hydrolysis probe in the qPCR/MCA or qPCR assays, respectively, helps to prevent false-negative detection due to insufficient extraction and/or amplification.

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Additional file 8:

TaqMan qPCR assay design for sensitive detection and quantification of A. astaci. Primers, but also TaqMan probe facilitate discrimination between A. astaci and various related or relevant oomycete species.

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