Characterization of a novelreal-time PCR assay.

Abstract

is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay forthat performs well under standard rtPCR conditions. Primers and probes were designed to target thegene. Reaction efficiencies for thegene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% ( ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (= 0.9784). The assay performed very well over a range of template input, from 10copies to the lower limit of quantification at 4 copies of thegenome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 otherspecies. To substantiate the high specificity of thegene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealedrather than. The complete protocol of the newly developedassay is provided to facilitate assay harmonization.