Development of a novel internal positive control for Taqman based assays

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.