SPUD qPCR assay confirms PREXCEL-Q softwares ability to avoid qPCR inhibition.
Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with about 15,500 SPUD amplicons yielded a Cq of 27.39 plus/minus 0.28 (at about 80.8% efficiency), while reactions spiked with about 7,750 SPUD plasmids yielded a Cq of 23.82 plus/minus 0.15 (at about 97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.
Department of Veterinary Pathology, Iowa State University, Ames, 50011-1250, USA. eag@iastate.edu <eag@iastate.edu>
J M
JM
Gallup
F B
FB
Sow
A
A
Van Geelen
M R
MR
Ackermann
D000818:Animals; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012756:Sheep; D012984:Software
D018799:Intercellular Adhesion Molecule-1