Comparative evaluation of real-time PCR (TaqMan®) with isolation for diagnosis of Phytophthora ramorum.

Kelvin Hughes, Ruth Griffin, Jenny Tomlinson, Neil Boonham, Victoria Barton, Patricia Giltrap, Ellie Hobden, Lynn Walker, Gilli Humphries, Ann Barnes, Paul Beales, Alan Inman & Charles Lane. Plant Health Group, Central Science Laboratory (CSL), York, UK. YO41 1LZ., k.hughes@csl.gov.uk, Tel +44 1904 462000

Field samples (323 in total) were tested for Phytophthora ramorum by isolation and CSL’s real-time PCR TaqMan® assay. The TaqMan® assay consists of reagents for specific amplification of P. ramorum DNA and a universal plant gene (cytochrome oxidase) as an internal reaction control. Samples were surface decontaminated and split into two equal parts. One was plated onto semi-selective agar (PARPH) and examined for P. ramorum after 6-days incubation, while DNA was extracted from the other and tested with the TaqMan® assay. Over 98 % of samples gave identical results for both isolation and PCR, with 25 of the 323 samples (8 %) testing positive for P. ramorum. Six samples (2%) did not agree however: three were isolation negative and PCR positive for P. ramorum, indicating that P. ramorum present was dead; two were isolation positive but PCR negative for P. ramorum and PCR positive for the internal control, thus showing no P. ramorum DNA was extracted from these samples, or it was below a PCR detectable level; and one sample was isolation positive and PCR negative for P. ramorum and the internal control, showing no amplifiable DNA was extracted from this sample. This trial demonstrated that isolation and TaqMan® PCR were equally reliable and robust for diagnosis of P. ramorum from the UK plant material tested.

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