Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification Using a Standardized Library of Isolates
Martin. F. N Coffey. M Hamelin. R Tooley. P Garbelotto. M Hughes. K Kubisiak. T
Phytopathology ; 2009 [Vol.99] Pages:390-403
Abstract
A number of molecular diagnostic procedures for detection of Phytophthora ramorum have been reported in the literature. In an effort to evaluate the specificity of 10 of these techniques a standardized DNA library for 317 isolates was assembled that included 60 described species as well as 22 taxonomically unclassified isolates. These were sent blind at a concentration of ca. 10 ng/µl (a concentration greater than would be encountered in field samples, but was used in an effort to fully evaluate specificity) to collaborators to evaluate the various diagnostic procedures. In general the procedures worked well with varying levels of specificity observed among the different techniques. Low levels of nonspecific amplification were observed for the mitochondrial markers and most of the real-time assays based on nuclear markers. The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not standalone and is used in conjunction with two other assays for diagnostic purposes. The results from the APHIS lab and another lab indicated that using three assays improved the accuracy of the results compared to looking at a single assay alone. The SSCP procedure accurately identified P. ramorum and was helpful in classification of other isolates to a species level. Given that one of the objectives of the trials was to determine if there would be any false positives, the DNA concentrations that were tested in all but one of the assays (6 to 10 ng/amplification) was higher than would be expected when processing field samples. As a result false positives for some of the assays (in particular real-time assays that had high Ct values) may not be representative of what might be encountered with field samples. Additional evaluations for these samples with a dilution series of target DNA are needed to evaluate specificity at DNA concentrations more reflective of what would be encountered in field assays. Trials evaluating marker performance with samples recovered from the field are in progress.
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