POSTERS: Pathogen detection, quantification and diagnosis
Development and validation of a TaqMan PCR method for rapid detection of Phytophthora fragariae in high volume root samples of strawberry
Daniel Bakker - Naktuinbouw. Sebastiaan Flanderhijn- Naktuinbouw, René Dekter- Naktuinbouw, Michel Ebskamp- Naktuinbouw
Phytophthora fragariae is the causal agent of red stele disease in strawberries. In the Netherlands, every year ~600 samples are collected from strawberry propagation companies to test for presence of P. fragariae. Samples consist of up to 200 gram of strawberry root tips. Until now samples are tested using the Duncan bait test. This test requires greenhouse space with controlled climate conditions and takes over five weeks to finish. In this project, we developed a root extract (RE) TaqMan PCR assay for detection of P. fragariae in large root samples as an alternative screening method for the bait test. DNA is purified using a magnetic bead based method and analyzed using TaqMan PCR. Quality of test results is monitored with a process control. The method is high throughput and results can be obtained within few days. In our validation study, we demonstrated that the RE TaqMan PCR was at least as sensitive as the bait test. The analytical specificity of the method, tested both in silico and in vivo, was high. The TaqMan PCR detected only P. fragariae from a panel of selected relevant Phytophthora spp.. Finally, based on repeatability and reproducibility, the method appears very robust. The root extract TaqMan PCR allows more cost-effective and rapid testing for P. fragariae in comparison with the traditional bait test. A method description and summary of the validation study of the P. fragariae RE TaqMan PCR assay will be presented.