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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGY AND EPIDEMIOLOGY OF EMERGING PLANT PATHOGENIC OOMYCETES Title: Development and Validation of a Tissue based Panel for the P. ramorum Proficiency Testing Program

item Mavrodieva, Vessela - APHIS
item Negi, S - APHIS
item Picton, D - APHIS
item Levy, Laurene - APHIS
item Tooley, Paul
item Shishkoff, Nina
item Luster, Douglas

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: April 30, 2008
Publication Date: August 15, 2008
Citation: Mavrodieva, V., Negi, S., Picton, D., Levy, L., Tooley, P.W., Shishkoff, N., Luster, D.G. 2008. Development and Validation of a Tissue based Panel for the P. ramorum Proficiency Testing Program. Phytopathology. 98:S100

Technical Abstract: Proficiency testing (PT) is a key element of a laboratory accreditation program. A tissue-based PT panel for the Sudden Oak Death pathogen, Phytophthora ramorum, used by the National Plant Protection Laboratory Accreditation Program (NPPLAP), was developed and validated in 2008 to assess proficiency of diagnosticians at critical stages of the diagnostic assay for P. ramorum. Healthy and P. ramorum infected Rhododendron 'Cunningham's White' leaves were used to prepare PT samples by lyophilization. Detached rhododendron leaves were dip-inoculated with 5000 sporangia per ml of P. ramorum and incubated in plastic moist chambers for 4-7 days at 20 degrees C in darkness. Each batch of lyophilized tissue was characterized by DNA extraction and real-time PCR (TaqMan) analysis of 5-10 percent of the PT panel samples. Mean (average) Ct values and standard deviation coefficients (STDV) were estimated for the P. ramorum (FAM) and plant DNA (TxRed) markers. To create PT tissue samples at varying concentrations, P. ramorum- infected tissue was diluted with healthy tissue at different ratios. The batch of 1:9 infected to healthy tissue ratios had a low STDV and produced a mean FAM Ct approximately 3.3 cycles higher then the undiluted P. ramorum infected batch. At greater ratios (1:99 and 1:999), STDVs were 3.42 and 3.96 respectively. These samples were not used for the panel. Alternatively, healthy plant tissue was spiked with P. ramorum culture DNA to produce low-level infection samples. Using this method we obtained a batch with a high mean FAM Ct and satisfactory STDV. Selected PT sample batches were then validated by three analysts to determine PT panel performance. PT panel stability was monitored monthly.

Last Modified: 11/27/2015
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