Exogenous Controls Increase Negative Call Veracity in Multiplexed, Quantitative PCR Assays for Phakopsora pachyrhizi

Quantitative polymerase chain reaction (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 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 a single-stranded oligonucleotide flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae primary assay but hybridizing to a unique fluorogenic probe; the third contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs neither qualitatively nor quantitatively affected their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and can be readily adapted to function in a variety of diagnostic Q-PCR assays; the plasmid 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.