Development of Virus-Mimicking Artificial Positive Controls for TaqMan® real-time PCR assays

TaqMan® real time PCR is the gold standard for virus detection, yet laboratories face challenges with acquiring positive controls, due to biosecurity restrictions and limited supply. Building on virus-mimicking artificial positive controls (ViMAPC) used in endpoint PCR, we have developed ViMAPCs for TaqMan® PCR. We utilize a sister virus taxon to create a positive control template which contains the target virus primer and probe sequences. As a proof-of-concept the novel strategy has been tested using both RNA and DNA viruses. ViMAPCs were tested in parallel with targeted virus-infected material with cross-verification tests carried out in a second laboratory and all met preset equivalence criteria. The technology provides highly reproducible positive controls for virus diagnostics allowing for the use of TaqMan® PCR when the procurement of true positive controls is difficult, such as a new incursion or biosecurity breaches.