Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.
Kansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA.
J Virol Methods. 2021 Oct;296:114220. doi: 10.1016/j.jviromet.2021.114220. Epub 2021 Jun 25.
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.
报道称，非典型猪瘟病毒（APPV）是导致新生仔猪 A 型 II 型先天性震颤的病原体。最初基于有限的序列信息设计的基于 PCR 的 APPV 诊断检测方法不能检测到大多数 APPV 株。一种敏感可靠的基于 PCR 的诊断检测方法对于准确检测 APPV 至关重要。在这项研究中，开发了一种定量逆转录 PCR（RT-qPCR）检测方法，用于可靠检测所有已知的 APPV 株。该检测方法的设计还包括猪 18S rRNA 基因作为内部对照，以监测 RNA 提取效率。克隆了 APPV 的两个基因片段，分别来自 NS5b 和 NS3，并用于确定检测的动态范围、线性和分析灵敏度/检测限（LOD）。单个和多重检测（双重和三重）的相关系数均>0.99，PCR 扩增效率均>90%。比较单个和多重检测的检测限和分析灵敏度表明，多重检测不会抑制 PCR 敏感性。基于分析灵敏度，APPV 靶标检测限为 7.75 个拷贝（NS5b）和 5.2 个拷贝（NS3）/反应。通过测试其他密切相关的瘟病毒和临床样本中其他常见猪病原体的核酸，验证了检测方法的特异性。还评估了最具差异的中国株的合成基因片段的检测灵敏度。检测 339 份已知的 APPV 阳性和 202 份阴性临床样本表明，该检测方法具有良好的诊断灵敏度和特异性。对 6 次独立运行的数据进行了评估，包括 3 个 Ct 范围的 3 个临床样本的 5 个重复，用于评估批内和批间重复性。该分析表明，三重检测的批内/批间变异系数分别为 0.71%和 0.01%，PCR 效率为 92.71%。检测 1785 份临床样本显示，美国猪群中 APPV 的流行率约为 19%，口腔液证明是一种可靠的病毒检测样本。这种多重 RT-qPCR 检测方法可快速可靠地检测猪群中的 APPV，是 APPV 监测和流行病学调查的有用工具。
