Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, PR China.
College of Biological and Pharmaceutical Engineering, West Anhui University, Lu'an 237012, PR China.
J Virol Methods. 2020 Nov;285:113963. doi: 10.1016/j.jviromet.2020.113963. Epub 2020 Aug 31.
Porcine circovirus 4 (PCV4) is a novel circovirus first discovered in China in April 2019. Here, we established an SYBR Green I-based real-time PCR for quantitative detection of PCV4. A pair of specific primers was designed based on the conserved region of Cap of PCV4. The standard curve of the established real-time PCR. assay showed a good linear relationship. The sensitivity of the established real-time PCR was 100 times greater than that of conventional PCR, and the detection limit of the assay was 3 × 10 copies. There was no cross-reactivity with other swine DNA viruses, showing good specificity. The intra-group variation coefficient was 0.37-0.78 %, and the inter-group variation coefficient was 0.57-0.94%, indicating that the assay has good repeatability. Moreover, the analysis of clinical samples showed that the positive detection rate of PCV4 was 10.71% (18/168), while that of conventional PCR was 8.93% (15/168). Interestingly, co-infection with PCV2 or PCV3, or both PCV2 and PCV3, was also detected. In conclusion, the established SYBR Green I-based real-time PCR may be a cost-effective and rapid method for PCV4 clinical diagnosis.
猪圆环病毒 4 型（PCV4）是一种新型圆环病毒，于 2019 年 4 月在中国首次发现。本研究建立了一种基于 SYBR Green I 的实时荧光定量 PCR 方法，用于 PCV4 的定量检测。根据 PCV4 衣壳蛋白 Cap 区保守序列设计了一对特异性引物。建立的实时荧光定量 PCR 标准曲线显示出良好的线性关系。该方法的灵敏度比常规 PCR 高 100 倍，检测限为 3×10 拷贝。与其他猪 DNA 病毒无交叉反应，具有良好的特异性。组内变异系数为 0.37-0.78%，组间变异系数为 0.57-0.94%，表明该方法具有良好的重复性。此外，对临床样本的分析表明，PCV4 的阳性检出率为 10.71%（18/168），而常规 PCR 的阳性检出率为 8.93%（15/168）。有趣的是，还检测到了 PCV2 或 PCV3 的混合感染，或 PCV2 和 PCV3 的双重感染。总之，建立的 SYBR Green I 实时荧光定量 PCR 可能是一种用于 PCV4 临床诊断的经济有效且快速的方法。
