Validation of a real-time PCR method for the detection of Phytophthora ramorum

• Published in: Bulletin OEPP Vol. 36; no. 2; pp. 409 - 414
• Main Authors: Chandelier, A., Ivors, K., Garbelotto, M., Zini, J., Laurent, F., Cavelier, M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.08.2006
• Subjects: Phytophthora; Pieris; Rhododendron; Viburnum
• ISSN: 0250-8052; 1365-2338
• DOI: 10.1111/j.1365-2338.2006.01020.x

To validate a real-time PCR method for the detection of Phytophthora ramorum, an intra-laboratory procedure was developed. The specificity of the TaqMan probe/primer sets was determined by carrying out real-time PCR on total DNA extracted from pure culture of several Phytophthora species. The limit of detection and the potential effects of plant substrates were evaluated by conducting the test on total DNA from healthy plant materials (Rhododendron spp., Viburnum spp. and Pieris spp.) spiked with known amounts of P. ramorum genomic DNA. The PCR efficiency was estimated through the linear regression of the dilution curve. Precision of the TaqMan assay was assessed on material from a single artificially infected plant (Rhododendron spp.). Two kinds of tissues were tested: a severely infected twig and an apparently healthy leaf. Intra-assay repeatability was evaluated on 10 replicates of the same DNA sample analysed in a single assay. Inter-assay reproducibility was evaluated on the same DNA sample amplified over five separate assays while the intersample reproducibility was evaluated on separate DNA extractions of four samples from both plant tissues amplified in a single assay.