Development of a quantitative real time RT-PCR assay for sensitive and rapid detection of emerging Atypical Porcine Pestivirus associated with congenital tremor in pigs.

Atypical Porcine Pestivirus (APPV) is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Initial PCR-based diagnostic tests to detect APPV were designed based on the limited sequence information and are not capable of detecting the majority of APPV strains. A sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. In this study, a quantitative reverse transcription PCR (RT-qPCR) assay was developed for reliable detection of all currently known APPV strains. The assay design also included swine 18S rRNA gene as an internal control to monitor RNA extraction efficiency. Two APPV gene fragments, one each from NS5b and NS3, were cloned and used to determine the dynamic range of detection, linearity and analytical sensitivity/limit of detection (LOD). Both individual and multiplex assays (duplex and triplex) had correlation coefficients of >0.99 and PCR amplification efficiencies of >90 %. Comparison of detection limit and analytical sensitivity between individual, and multiplex assays indicated no inhibition of PCR sensitivity upon multiplexing. The detection limit for APPV target, based on analytical sensitivity, is 7.75 copies (NS5b) and 5.2 copies (NS3) per reaction. Assay specificity was verified by testing nucleic acids of other closely related pestiviruses and clinical samples that are positive for other common swine pathogens. Assay sensitivity was also assessed on synthesized gene fragments of the most divergent China strains. Testing 339 known APPV-positive and 202 negative clinical samples demonstrated a good diagnostic sensitivity and specificity. Data from six independent runs, including 5 replicates of three clinical samples with three Ct ranges, were utilized to assess inter-assay repeatability and intra-assay reproducibility. This analysis demonstrated intra-assay/inter-assay coefficients of variation of 0.71 % and 0.01 %, respectively, with a PCR efficiency of 92.71 % for the triplex assay. Testing of 1785 clinical samples revealed ∼19 % prevalence of APPV in the US swine herds and oral fluids demonstrates to be a reliable specimen for viral detection. This multiplex RT-qPCR assay offers a rapid and reliable detection of APPV in swine herds and serves as useful tool in APPV surveillance and epidemiological investigations.