Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100176, China.
Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, 100176, China.
Mol Cell Probes. 2019 Feb;43:50-57. doi: 10.1016/j.mcp.2018.11.005. Epub 2018 Nov 20.
Porcine circovirus 3 (PCV3), a newly emerged circovirus, is associated with porcine dermatitis and nephropathy syndrome, reproductive failure and multi-systemic inflammation disease, and is widely distributed in pig populations worldwide. Therefore, developing specific diagnostic assays will be important for controlling this emerging pathogen. In this study, we developed a novel droplet digital PCR (ddPCR) assay targeting the PCV3 cap gene to improve the sensitivity of PCV3 detection. The established assay is highly specific to PCV3, and does not cross react with other important swine pathogens. The assay's detection limit was 1.68 ± 0.29 copies of PCV3 DNA per reaction (n = 8), an approximately 10-fold greater sensitivity than that of our previously developed quantitative real-time PCR (qPCR) assay for the same virus. The ddPCR assay results were highly reproducible, with intra- and inter-assay coefficient of variation values of <9.0%. Of the 239 archived pig tissue and serum samples, 42 tested positive for PCV3 by the ddPCR assay. Among the 42 positive samples, 31 tested positive by the qPCR assay. Notably, PCV3 was detected in the serum samples collected from commercially imported healthy boars from the US, France and the UK during 2011-2017. The overall agreement between the two assays was 95.39% (228/239). Furthermore, the linear regression analysis showed that the ddPCR and the qPCR results were significantly correlated with an R value of 0.9945. Collectively, these results indicate that the ddPCR assay is a robust diagnostic tool for sensitive detection of PCV3, even in samples with low viral loads.
猪圆环病毒 3 型（PCV3）是一种新出现的圆环病毒，与猪皮炎肾病综合征、繁殖失败和多系统炎症疾病有关，广泛分布于世界各地的猪群中。因此，开发特定的诊断检测方法对于控制这种新出现的病原体非常重要。在本研究中，我们针对 PCV3 的 cap 基因开发了一种新的液滴数字 PCR（ddPCR）检测方法，以提高 PCV3 的检测灵敏度。该方法对 PCV3 具有高度特异性，不会与其他重要的猪病原体发生交叉反应。该方法的检测限为每个反应 1.68±0.29 拷贝的 PCV3 DNA（n=8），比我们之前开发的针对同一病毒的定量实时 PCR（qPCR）检测方法的灵敏度高约 10 倍。ddPCR 检测方法的结果具有高度可重复性，内和间试验变异系数值均<9.0%。在 239 份存档的猪组织和血清样本中，42 份样本经 ddPCR 检测为 PCV3 阳性。在 42 份阳性样本中，31 份样本经 qPCR 检测为阳性。值得注意的是，在 2011 年至 2017 年期间，从美国、法国和英国商业进口的健康公猪的血清样本中检测到了 PCV3。两种检测方法的总符合率为 95.39%（228/239）。此外，线性回归分析表明，ddPCR 和 qPCR 结果具有显著相关性，R 值为 0.9945。综上所述，这些结果表明，ddPCR 检测方法是一种用于灵敏检测 PCV3 的可靠诊断工具，即使在病毒载量较低的样本中也是如此。
