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Title: Adaptation of a Phytophthora ramorum Real-Time Polymerase Chain Reaction Assay based on a mitochondrial gene region for use on the Cepheid SmartCycler

Author
item Sechler, Karen
item Carras, Marie
item Shishkoff, Nina
item Tooley, Paul

Submitted to: Plant Health Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2009
Publication Date: 2/13/2010
Citation: Sechler, K.E., Carras, M.M., Shishkoff, N., Tooley, P.W. 2010. Adaptation of a Phytophthora ramorum Real-Time Polymerase Chain Reaction Assay based on a mitochondrial gene region for use on the Cepheid SmartCycler. Plant Health Progress. 2010 - 0212-01-RS.

Interpretive Summary: It is important that Phytophthora ramorum, the organism that has caused the deaths of thousands of oak trees in California and Oregon, not spread beyond the regions already infected. Quarantine regulations to restrict the spread require that nurseries shipping plants outside the infested area be inspected and declared free of the pathogen. The U.S. Forest Service and many plant pathology extension labs perform inspections of nurseries outside the infested area to confirm that the organism has not spread. Since P. ramorum can be difficult to detect, it is essential that as many inexpensive, easy and reliable detection methods be made available as is possible. This research adapted and optimized a previously described PCR assay for use with a PCR platform available in many diagnostic labs across the country. The detection limit using this new platform was determined to be accurate to very, very small numbers (one quadrillionth of a gram). No cross-reactivity was observed for the seven additional Phytophthora species tested, which included species known to cross-react in other tests and the recently discovered Phytophthora foliorum and Phytophthora kernoviae. These facts indicate that this method would be reliable for detecting the pathogen.

Technical Abstract: Detection of Phytophthora ramorum (causal agent of sudden oak death) in U.S. commercial nurseries has led to quarantine regulations including inspection of nurseries in infested areas. Since P. ramorum can be difficult to culture from symptomatic tissue, methods such as real-time PCR provide rapid and reliable detection. This research adapted and optimized a previously described mitochondrial based TaqMan assay for use with a Cepheid SmartCycler v.1 and ready-to-use lyophilized PCR beads. Although variable detection of DNA was possible at or below 1 fg, the detection limit on the SmartCycler was determined to be at 10 fg, a 10-fold increase over the limit on the ABI platform. Quantification estimates based on data from both the ABI Prism 7700 Sequence Detector System and the Cepheid SmartCycler v.1 were similar, but cycle threshold (Ct) values from the Cepheid instrument were several cycles lower than those observed on the ABI instrument. No cross-reactivity was observed on the SmartCycler for the seven additional Phytophthora species tested, which included species known to cross-react in other tests and the recently discovered Phytophthora foliorum and Phytophthora kernoviae.