Validation of a real-time PCR method for the detection of phytophthora ramorum
A. CHANDELIER, J. ZINI , F. LAURENT & M. CAVELIER
Walloon Agricultural Research Centre, Department Biological Control and Plant Genetic
Resources Rue de Liroux, 4 ; B-5030 Gembloux, Belgium. chandelier@cra.wallonie.be
Since 2003, our laboratory has participated to the survey set up by the Belgian Plant Protection
Service for the detection of Phytophthora ramorum in ornamental nurseries and parks. The pathogen
is detected by isolation on selective medium (De Merlier et al, 2005) and by real-time PCR using a
method developed by Ivors and Garbelotto (2002).
The molecular test consists of two steps : a DNA extraction from plant material (leave, twig or stem
base) ground in liquid nitrogen (using a commercial kit) and a PCR amplification with a Taqman®probe.
To validate the molecular method, an intra-laboratory validation procedure based on different
standards (ISO 22174 and XP V 03-020-2) was developed in a context of accreditation (ISO/IEC
17025, general requirements for the competence of testing and calibration laboratories). Various
parameters were studied. The specificity was determined by carrying out real-time PCR on total DNA
extracted from pure culture of several Phytophthora species. The limit of detection, the sensitivity and
the linear range were evaluated by conducting real-time PCR on total DNA from healthy plant spiked
with known amounts of P. ramorum DNA. The PCR efficiency was estimated through the linear
regression of the dilution curve (Ct values as a function of the target DNA content). Precision of the
Taqman® assay was assessed on material from a single infected plant (Rhododendron). Intra-assay
repeatability was evaluated on ten replicates of the same DNA sample analysed in a single assay.
Inter-assay-reproducibility was evaluated on the same DNA sample amplified over five separate
assays while the inter-sample reproducibility was evaluated on separate DNA extractions of eight
samples from the same infected plant amplified in a single assay.
De Merlier D, Chandelier A, Debruxelles N, Noldus M, Laurent F, Dufays E, Claesens H, Cavelier M
(2005) Characterisation of Alder Phytophthora isolates from Wallonia and development of SCAR
primers for their specific detection. Journal of Phytopathology 153, 99-107.
Ivors K & Garbelotto M (2002) Taqman PCR for detection of Phytophthora DNA in environmental
plant samples. In Proceedings of “Sudden Oak Death, a science Symposium”, December 2002, pp. 56.
Monterey, CA (USA)