Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

Abstract

Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.

Simultaneous Detection and Quantification of <i>Phytophthora nicotianae</i> and <i>P. cactorum</i>, and Distribution Analyses in Strawberry Greenhouses by Duplex Real-time PCR

“…Diagnostic PCR methods and specific primers have been developed for Phytophthora species including P. nicotianae (12, 18, 22, 28) and P. cactorum (2, 5, 21, 30), but most of these studies aimed at the detection of a single pathogen. Multiplex PCR assays allow the simultaneous detection of several species, and facilitate large-scale sample processing (25); however, multiplex PCR has been applied rarely in plant pathology (13, 14, 24, 31, 34, 35). This is partially due to the difficulties related to the development of quantitative multiplex assays and to the reduced sensitivity of multiplex PCR compared with simplex PCR (31).…”

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

“…Some aspects of our existing TaqMan method (33), such as the run time and the complexity of the equipment required, are not ideally suited to on-site testing. The assay on which this work is based was itself adapted from a laboratory TaqMan assay (12) which is better suited to large-scale use in a routine diagnostic setting. The original assay uses "universal" thermal cycling conditions which are often used for TaqMan real-time PCR (many TaqMan primers and probes are designed to have melting temperatures which allow universal cycling conditions to be used).…”

“…P. ramorum-specific primers (Pram-114F and Pram-190R) and a TaqMan probe (Pram probe), designed based on the ITS sequence of a range of Phytophthora spp. as previously described (12), were used as the basis for the design of a duplex scorpion primer, molecular beacon, and alternative reverse primer (Pram-199R) (Fig. 1).…”

“…In particular, real-time PCR methods have the advantages of speed, accuracy, and sensitivity over other methods (7,28,30,43) and can be closedtube systems involving no postamplification steps. 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).…”

Fifty years of oomycetes—from consolidation to evolutionary and genomic exploration

“…Many PCR assays were developed for P. ramorum (e.g. Tomlinson et al 2007;Bilodeau et al 2007;Tooley et al 2006;Martin et al 2004;Hughes et al 2006;Hayden et al 2006), to the point of causing some confusion in the international regulatory community as to which one should be routinely used. The international ring trial to evaluate several of these methods simultaneously with the same samples should become a model for other pathogens ).…”