Exogeneous controls to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

Authors: HAUDENSHIELD, JAMES - University Of Illinois; Hartman, Glen

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2010
Publication Date: 3/1/2011
Citation: Haudenshield, J.S., Hartman, G.L. 2011. Exogeneous controls to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi. Plant Disease. 95:343-352.

Interpretive Summary: Soybean rust, caused by Phakopsora pachyrhizi, is a destructive foliar disease of soybean. Quantitative PCR (Q-PCR) is a molecular tool to detect DNA, and can be used to detect different pathogens including the one that causes soybean rust. Because of the extreme sensitivity of Q-PCR, the DNA of single spores of these fungi 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. Three synthetic DNA molecules of artificial and arbitrary sequence were constructed as internal controls to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. These internal controls contain no appreciable similarity to natural sequences present in public databases. With the sequence information provided, an appropriate internal control assay to validate true-negative results and detect false-negative calls may be devised for a wide array of Q-PCR applications in minutes, and at relatively low cost. This information will be used by molecular biologist, plant pathologist, and other scientists interested in using internal controls with their molecular detection assays.

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 and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, the DNA of single urediniospores of these fungi 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. Three synthetic DNA molecules of artificial and arbitrary sequence were constructed as multiplexed internal controls (ICs) to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. The first two, PpaIC and PmeIC, are single-stranded oligonucleotides flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae assay, but hybridizing to a unique fluorogenic probe; the third, HHIC, contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs contain no appreciable similarity to natural sequences present in public databases, and were found to neither qualitatively nor quantitatively affect their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and HHIC was found to successfully validate true negatives in similar Q-PCR assays for other soybean pathogens, as well as to function as a tracer molecule during DNA extraction and recovery.