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Introduction
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Real time quantitative PCR is a new approach in PCR-technology. It is much faster and more reliable than other quantitative PCR-techniques such as competitive PCR and picture-analysis after geleletrophoresis. The challenge of our project was to develop new assays for the quantification of mycorrhizal, leaf and root pathogens using real time quantitative PCR.
Part 1 Principle of real time quantitative PCR
Part 2 Development of specific PCR primers and fluorogenic probes
Part 3 Real time quantification of fungal biomass
Part 4 DNA extraction from infested beech roots
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Conclusions
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The presented real time quantitative PCR assays together with an reliable DNA-extraction method and the specific PCR primers for the different pathogens enable the different working groups of the SFB 607 to quantify mycorrhizial and plant pathogenic fungi. With the knowledge of this project other TaqMan-assays were developed by A 9, B 7 and B 8.
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Summary
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Specific primers and dual-labelled flourogenic probes were designed for polymerase chain reaction (PCR)-based detection of both, mycorrhizal and pathogen DNA. Based on the on-line connection with an automated ABI Prism 7700 sequence detector, amplicon quantification was directly performed during the PCR. The starting copy numbers of target sequences present in each reaction were calculated by comparing the Ct-values of unknown samples to the Ct-values of standards with known amounts of DNA. The Ct-value depends on the input of starting copies and is defined as that cycle number at which a statistically significant increase in the reporter fluorescence can first be detected. DNA was extracted from 11 as well as 100 spores of the mycorrhizal fungus Glomus mosseae and quantified by using the fluorescent PCR technology. Furthermore, DNA of Phytophthora infestans, causal agent of late blight of potatoes, was quantified after extraction form artificially infected potato tubers and naturally infected field plants. Phytophthora citricola DNA, causing root-rot deseases, was quantified after isolation from artificially inoculated seedling roots of beech and oak. The results demonstrate that novel real-time PCR techniques are a powerful universal tool in modern phytopathological research.
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