TY - JOUR
T1 - Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA)
AU - Davari, M.
AU - van Diepeningen, A.D.
AU - Babai-Ahari, A.
AU - Arzanlou, M.
AU - Najafzadeh, M.J.
AU - van der Lee, T.A.J.
AU - de Hoog, G.S.
N1 - Reporting year: 2012
PY - 2012
Y1 - 2012
N2 - Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-alpha (EF-1 alpha) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated what or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals. (C) 2012 Elsevier B.V. All rights reserved.
AB - Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-alpha (EF-1 alpha) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated what or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals. (C) 2012 Elsevier B.V. All rights reserved.
U2 - 10.1016/j.mimet.2012.01.017
DO - 10.1016/j.mimet.2012.01.017
M3 - Article
SN - 0167-7012
VL - 89
SP - 63
EP - 70
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 1
ER -