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Int J Mol Sci. 2008 Nov;9(11):2306-21. doi: 10.3390/ijms9112306. Epub 2008 Nov 25.

Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error.

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1
University of Göttingen, Department of Crop Sciences, Germany.

Abstract

Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5-1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5-1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed.

KEYWORDS:

DNA extraction; Fusarium culmorum W.G. Smith; Fusarium graminearum Schwabe; Fusarium head blight; real-time PCR; sampling error

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