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refereed
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Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison.

GND
1058930834
Affiliation
Julius Kühn-Institute (JKI), Institute for Epidemiology and Pathogen Diagnostics, Germany
Sedaghatjoo, Somayyeh;
Affiliation
Bavarian State Research Center for Agriculture, Institute for Crop Science and Plant Breeding, Germany
Forster, Monika K.;
Affiliation
Technical University of Munich, TUM School of Life Sciences, Chair of Technical Microbiology, Germany
Niessen, Ludwig;
Affiliation
University of Goettingen, Molecular Phytopathology and Mycotoxin Research, Germany
Karlovsky, Petr;
Affiliation
Bavarian State Research Center for Agriculture, Institute for Crop Science and Plant Breeding, Germany
Killermann, Berta;
GND
12412514X
Affiliation
Julius Kühn-Institute (JKI), Institute for Epidemiology and Pathogen Diagnostics, Germany
Maier, Wolfgang

Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

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