Poster: Diseases of Plants: Disease Detection & Diagnosis
Extracting DNA for qPCR amplification from plant tissue in ELISA buffer
N. OSTERBAUER (1), S. Navarro (2) (1) Oregon Dept. of Agriculture, U.S.A.; (2) Oregon Dept. of Forestry, U.S.A.
USDA APHIS mandates testing protocols for Phytophthora ramorum. Diagnostic labs use an ELISA kit to screen for Phytophthora infection in leaves. If a leaf tests ELISA-positive, a second sample is collected from the leaf for DNA extraction and molecular testing. This may delay detection by a day. DNA extraction directly from macerated leaf tissue in ELISA buffer would speed testing. To determine if DNA extracted from such tissue would be adequate for molecular testing, 198 leaf samples from a nursery were tested with ELISA, with 30 testing positive. For each ELISA-positive sample, leaf tissue was collected and DNA extracted following WI-B-T-2-3 Rev. 1; DNA was also extracted from the macerated leaf tissue in ELISA buffer. Tissue-DNA and ELISA-DNA was then tested for P. ramorum following N-R-WI-008-0 using the reporter dyes FAM (P. ramorum-specific) and JOE (internal control); all DNA was tested undiluted and diluted 1:10. For all samples, the tissue-DNA and ELISA-DNA tested negative for P. ramorum (FAM Cq = 0.00) at both dilutions. For the internal control, the tissue-DNA tested positive for amplifiable DNA with a mean Cq of 27.30, whereas the ELISA-DNA tested positive with a mean Cq of 26.41. For diluted DNA, the mean Cq for tissue-DNA was 20.45 and for ELISA-DNA was 20.79. The results suggest amplification of the internal control was comparable between DNA extracted from leaf tissue and DNA extracted from macerated leaf tissue in ELISA buffer.