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TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods

The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5′ fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.