On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

Abstract

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a costeffective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.Phytophthora ramorum is the causal agent of extensive oak mortality (commonly known as sudden oak death) in coastal forests in California (27) and southern Oregon (12,25). This pathogen also causes ramorum leaf blight and dieback on a range of other plant species (9) and can have a profound and devastating effect on forest ecosystems. A distinct population of the same pathogen (6, 35) is found in a number of European countries (10,20,23,37), mostly causing dieback and leaf blight on a range of ornamental plants in nurseries and landscaped areas (2). There have also recently been a number of incidences of lethal bark cankers caused by P. ramorum in native and nonnative trees in Europe (7). P. ramorum has a broad and expanding host range (3,4,10,11,19,20,28,31), and as a result of the threat posed to forest ecosystems, the movement of a variety of its host species is subject to restrictions in Europe and the United States. Emergency European Community phytosanitary measures for P. ramorum were introduced in 2002 (1), and in the United States quarantine restrictions at both the federal and state levels control the movement of a variety of plant species from infested areas in California and Oregon (31). The availability of rapid and accurate detection methods for P. ramorum is critical to allow its prevalence to be monitored and to expedite management or eradication steps to prevent its introduction and minimize its spread.The identification of P. ramorum is not possible based on host symptoms alone due to the considerable variation in their expression and because a range of other causes can produce similar symptoms. These include infections by several other Phyt...

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

“…These assays may have a number of limitations which could restrict their use in the field. For instance, many TaqMan assays use standard thermal cycling conditions, but even a rapid cycling real-time PCR instrument such as the Smart Cycler II (Cepheid, Sunnyvale, CA) takes over an hour to complete 40 cycles of standard TaqMan thermal cycling (33). This amount of time may be prohibitive when carrying out testing in the field.…”

“…Previously described assays for P. ramorum designed within the internal transcribed spacer 1 (ITS 1) region of the nuclear ribosomal DNA have shown cross-reactions with high concentrations of DNA extracted from Phytophthora lateralis (9,33), which differs from P. ramorum by only 11 bp in the ITS region (9). Cross-reactivity has been observed at only high DNA concentrations (more than 1 ng P. lateralis DNA in the PCR), and material being tested for P. ramorum is very unlikely to contain P. lateralis (a root pathogen).…”

“…TaqMan (11,16) is the most widely used real-time PCR system, and assays using this detection chemistry have been described for a range of plant pathogens (3,4,14,39,38,40), including assays for the detection of Phytophthora ramorum both in the laboratory (8,12,34) and in the field (33). A single round of real-time PCR has been found to be sufficiently sensitive to achieve the detection of 10 to 100 fg P. ramorum DNA in plant material (8,33). However, nested PCR has been used to improve the sensitivity of detection in some matrices (8).…”

A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

“…The time required for preparing the dry reagents consists of approximately 30 minutes of hands-on time for preparing the reagents and dispensing them into the reaction vessels and of overnight drying after which the reagents are ready to use or can be packaged for storage. With BSA as a stabilizing agent the stability of the dried reagents was not sufficient and therefore we tested trehalose that has been shown to protect dried reagents [10] as well as act as a PCR enhancer [16,18]. The reagents air-dried together with 0.2 M trehalose with separately dried DNA-polymerase were shown to be stable at least up to eight weeks of storage at room temperature.…”

Proceedings of the sudden oak death third science symposium

“…The CSL protocol was developed as a real-time P. ramorum specific PCR assay (Tomlinson andothers 2005, Hughes 2003). Phytophthora ramorum specific primers and labeled probe, along with primers and probe designed to amplify plant DNA, are run together.…”