Use of real-time PCR for quantitative analysis of pathogen of cereals in primary food sources
2005
Gubis, J.(Vyskumny ustav rastlinnej vyroby, Piestany (Slovak Republic) | Hudcovicova, M.
The objective of this paper was to input real-time PCR quantification of pathogen cereals into research area of plant pathology in Slovakia. A real-time quantitative PCR technique has been used as a rapid and sensitive seed health test for P. teres, R. secalis and S. tritici from artificially and field infected seeds. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seeds can be quantified to the picogram level. In these experiments pathogen-specific primers for R. secalis and S. tritici were developed too. Specific primer set for R. secalis (RS17F and RS17R) was derived from ITS regions of rDNA of this pathogen and primer set for S. tritici (STRT1F and STRT1R) was derived from beta-tubuline gene of this pathogen. It has been proved that primers specific to P. teres, R. secalis and S. tritici can reliably diagnose pathogen DNA as well as its presence in the mixture with barley or wheat DNA. DNA from infected seeds was isolated by the Adgen DNA Extraction System (Adgen Ltd.) and Wizard DNA Clen-Up System (Promega).
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