Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 4/11/2008
Publication Date: 6/1/2008
Citation: Haudenshield, J.S., Hartman, G.L. 2008. Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi [abstract]. Phytopathology. 98:S66.
Technical Abstract: Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single urediniospore of this fungus can be detected from total DNA extracts of environmental samples. However, some DNA preparations unpredictably contain PCR inhibitors that increase the frequency of false-negatives indistinguishable from true-negatives. Two synthetic DNA molecules of artificial and arbitrary sequence were constructed, and their functionality demonstrated with the matching primers and probes as multiplexed, internal controls (ICs), to cull false-negative results by producing a positive signal that validates the PCR process within each individual reaction. The first, PpaIC, is a single-stranded oligonucleotide flanked by sequences complementary to the primers of the P. pachyrhizi assay itself, but targeted by a unique probe. The second, HHIC, is a dsDNA designed to utilize unique primers and probe, and was prepared as a cloned sequence in a linearized plasmid. Either IC may be added in trace amounts to raw samples before DNA extraction, or to reagent mixtures during assay set-up. Neither IC has significant similarity to natural sequences present in public databases and, whereas PpaIC will be useful mainly in P. pachyrhizi testing, HHIC has potential for use in a broad range of multiplexed Q-PCR assays when a natural internal control against false-negatives is unavailable.