Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique
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Phytophthora kernoviae is a recently described pathogen causing leaf blight, aerial dieback and bleeding cankers on trees and shrubs in parts of Great Britain and Ireland and recently reported in New Zealand. This paper describes the development of a TaqMan real-time PCR assay based on internal transcribed spacer (ITS) sequence to aid diagnosis of this pathogen in culture and in plant material. The assay showed no cross reaction with 29 other Phytophthora species, including the closely related species P. boehmeriae, and detected at least 1.2 pg of P. kernoviae DNA per reaction. A rapid and simple method can be used to extract DNA prior to testing by real-time PCR, and a plant internal control assay can be used to aid interpretation of negative results. A comparison of real-time PCR and plating for 526 plant samples collected in the UK indicated that this assay is suitable for use in routine screening for P. kernoviae.
KeywordsDiagnostic sensitivity Diagnostic specificity Plant health Rhododendron
Funding for this work was provided by Plant Health Division of Defra and work was performed under license number PHL 251A/4736 (03/20004). The authors would like to thank Dr Paul Beales and Dr Rebecca Weekes for their invaluable support and guidance.
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