The production of healthy seed and plant material is a fundamental prerequisite for the establishment of ecologically stable and economically productive forest stands. Nevertheless, as in the past, forest plant production is threatened by biotic harmful factors and new, invasive species are becoming increasingly important as a result of climate change and globalization.
DNA-based methods have significantly accelerated the detection of plant pathogens, but are time-consuming, costly and require extensive equipment. Loop initiated isothermal amplification (LAMP) is an efficient and cost-effective alternative to the classical polymerase chain reaction (PCR). The reaction takes place as a one-step assay at a constant temperature and can be evaluated visually.
In the project “TreeLAMP”, a LAMP method is established for Rhabdocline pseudotsugae, one of the most important needle pathogens of Douglas fir. To date, 32 LAMP primer sets have been derived from the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and tested. The results showed clear differences between the primer sets both in terms of reaction rate and concentration of the amplified products. Following extensive work to optimize the LAMP reaction, a method is now available that allows the reliable detection of R. pseudotsugae at a constant temperature (65°C) and a reaction time of 1.5 hours. The detection limit is currently at 0.02 pg/µl R. pseudotsugae DNA. The current focus of the project is the optimisation of DNA extraction. In addition to conventional DNA kits, methods adapted to the detection procedure are used, which allow DNA extraction to be carried out quickly and without great technical effort.
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