Real-time PCR method and its application for the detection of fungi contaminats in barley
2006
Gubis, J.(Vyskumny ustav rastlinnej vyroby, Piestany (Slovak Republic) | Hudcovicova, M. | Cervena, V. | Bojnanska, K. | Kraic, J.
The aim of this work was to detect and qualify barley pathogens Pyrenophora teres and Rhynchosporium secalis in seed samples. Real-time PCR technique was used as a fast and sensitive quantitative method of testing the health state of naturally infected seed. By using the fluorescent reporter dye SYBR GreenI for real-time PCR the pathogen DNA extracted from the infected seed can be quantified already on the picogram level. DNA from the infected seed was isolated using the Adgen DNA Extraction System (Adgen Ltd.) and the Wizard DNA Clean-Up system (Promega). Primers for RT-PCR were in the case of P. teres taken over from the literature and in the case of R. secalis a new pathogen-specific primer RS17F-RS17R was designed; at first its specificity was proved using several pathogen species in the PCR reaction. After the optimalization of the primers concentration, reaction mixture and other parameters of RT-PCR the pathogen DNA of P. teres and R. secalis were quantified in naturally infected seed samples. The results obtained in our conditions link to the previous work and can be utilized in laboratory detection of the seed-borne fungi pathogens of barley, which enables the accurate and early diagnosing and effective control of the pathogens quantity in primary food sources.
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