|De Cook, A.w.a.m.|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/22/2006
Publication Date: 5/1/2007
Citation: Bilodeau, G.J., Levesque, C.A., De Cook, A., Duchaine, C., Kristjansson, G., Uribe, P., Martin, F.N., Hamelin, R.C. 2007. Molecular detection of phytophthora ramorum by real-time pcr using taqman, sybr green and molecular beacons with three genes. Phytopathology 97:632-642. DOI:10.1094/PHYTO-97-5-0632 Interpretive Summary: The objective of this study was to develop molecular diagnostic techniques for detection of Phytophthora ramorum, the pathogen responsible for causing sudden oak death. Three different regions of the nuclear genome were used to construct species-specific markers and three different real-time PCR technologies were evaluated for sensitivity and specificity. The TaqMan system worked the best with the markers made for the ITS region, beta tubulin gene and the elicitin gene working equally well.
Technical Abstract: Sudden oak death, caused by Phytophthora ramorum, is a severe disease that can affect many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to not infested areas. Molecular assays have been developed to rapidly detect and identify P. ramorum but one difficulty encountered with some of these assays is the inability to reliably distinguish between P. ramorum and closely related species. In order to overcome potential cross reaction, beta-tubulin and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Real-time PCR assays were designed using existing internal transcribed spacer (ITS) sequences as well as the new beta-tubulin and elicitin sequences. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR® Green. Primers specific to P. ramorum were designed to amplify a fragment of beta-tubulin and were used in real-time PCR assays in conjunction with the three technologies to detect this pathogen. The assays and primers differentiated P. ramorum from the 65 species of Phytophthora tested, including P. lateralis. The assays based on detection of the ITS and elicitin tended to have lower Ct values than those using beta-tubulin and seem to be more sensitive. The assays developed were also used with DNA extracts from 49 infected and uninfected plant samples. No false negatives and no false positives were observed using plant material with the false positives were observed using plant material with the assays based on beta-tubulin and elicitin. Overall, TaqMan assays with ITS or elicitin were found to have the best combination of sensitivity and specificity.