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

2021 | journal article. A publication with affiliation to the University of Göttingen.

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​Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison​
Sedaghatjoo, S.; Forster, M. K.; Niessen, L.; Karlovsky, P. ; Killermann, B. & Maier, W.​ (2021) 
Scientific Reports11(1) art. 11611​.​ DOI: https://doi.org/10.1038/s41598-021-91098-2 

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Authors
Sedaghatjoo, Somayyeh; Forster, Monika K.; Niessen, Ludwig; Karlovsky, Petr ; Killermann, Berta; Maier, Wolfgang
Abstract
Abstract 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.
Abstract 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.
Issue Date
2021
Journal
Scientific Reports 
Organization
Fakultät für Agrarwissenschaften ; Department für Nutzpflanzenwissenschaften ; Abteilung Molekulare Phytopathologie und Mykotoxinforschung 
eISSN
2045-2322
Language
English

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