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ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples

Niessen, Ludwig, Gräfenhan, Tom, Vogel, Rudi F.
International journal of food microbiology 2012 v.158 no.3 pp. 171-185
DNA primers, wheat, genes, ATP citrate synthase, Fusarium acuminatum, cross reaction, barley, Fusarium tricinctum, detection limit, food analysis, household equipment, fluorescence, DNA, microwave ovens, washing, polymerase chain reaction, yeasts, hybridization, adenosine triphosphate, Fusarium avenaceum, heating systems, nucleotide sequences
The combined data set of the acl1 and tef-1α gene sequences of 61 fungal strains assigned to Fusarium tricinctum, Fusarium avenaceum, Fusarium acuminatum, Fusarium arthrosporioides, Fusarium flocciferum and Fusarium torulosum were used to study the phylogenetic relations between taxa. F. tricinctum, F. acuminatum and F. avenaceum formed distinct clades. Members of the F. tricinctum/F. acuminatum clade fall into three well supported lineages, of which the largest includes the epitype of F. tricinctum. Loop-mediated isothermal amplification (LAMP) was used to amplify a 167bp portion of the acl1 gene in F. tricinctum (Corda) Saccardo. DNA amplification was detected in-tube by indirect calcein fluorescence under black light after 60min of incubation at 65.5°C. The assay had a detection limit of 0.95pg of purified genomic DNA of F. tricinctum CBS 410.86 per reaction, corresponding to ca. 18 genomic copies of the acl1 gene. Specificity of the assay was tested using purified DNA from 67 species and subspecies of Fusarium as well as 50 species comprising 22 genera of other filamentous fungi and yeasts. The assay detected 21 of the 23 F. tricinctum strains tested. Cross reactivity was observed with eight out of 13 strains in F. acuminatum but with none of 17 F. avenaceum strains tested. Specificity was further confirmed by conventional PCR with primers designed from the same gene. Detection of F. tricinctum from culture scrapings directly added to the reaction master mix, in DNA extracts from wheat, in single barley grains or in washings of bulk grain samples are proposed as possible applications showing the suitability of the method for food analysis. Finally it was demonstrated that the LAMP reaction can be run using simple lab equipment such as a heating block, water bath, hybridization oven or household equipment, e.g. a microwave oven.