Head-to-head comparisons of sensitivity and specificity among 5 real-time PCR assays diagnostic for Phytophthora ramorum

• Published in: Phytopathology Vol. 98; no. 6; p. S179
• Main Authors: Zeller, KA, DeVries, R M, Levy, L
• Format: Journal Article
• Language: English
• Published: 01-06-2008
• Subjects: Phytophthora
• ISSN: 0031-949X

In response to Stakeholder requests, we adapted and validated five alternative Real-time PCR diagnostic assays for Phytophthora ramorum developed by other laboratories that target DNA loci in the ribosomal repeats, mitochondrial DNA, and in individual single-copy genetic loci for use in the Cepheid Smartcycler. We have compared relative sensitivity and specificity of the methods by testing on a common set of >130 previously diagnosed environmental samples. Three of the 5 tested diagnostic methods failed to cross-react with DNAs from closely related Phytophthora foliorum, P. hibernalis or P. lateralis. However, each of these three methods also displayed lower sensitivity for target DNA of P. ramorum than do the validated conventional nested PCR assay and ITS-targeting Real-time PCR assay. Two of the three methods (mitochondrial Cox locus, and genomic Elicitin locus) are able to reliably detect P. ramorum DNAs to >50fg of target DNA. When combined with the currently used ITS-targeting Real-time PCR assay, either of these two Real-time PCRs should reduce the frequency of false positive results on initial tests of environmental samples for P. ramorum. The current ITS and Elicitin Real-time assays are multiplexed with internal control amplicons for mitochondrial and genomic DNAs, respectively, that replicate the internal controls in the current diagnostic system that make this combination desirable.