Chen Wanting, Jiang Dike, Xiao Lu, Zhang Pengfei, Luo Yan, Yang Zexiao, Yao Xueping, Wang Yin, Wu Xulong
Chengdu Agricultural College, Chengdu 611130, China.
College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China.
J Vet Res. 2022 Mar 25;66(1):29-33. doi: 10.2478/jvetres-2022-0004. eCollection 2022 Mar.
Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims.
Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus' genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established.
The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%).
This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.
猪圆环病毒4型（PCV4）于2019年首次在中国湖南省一群患有猪呼吸道疾病、皮炎和肾病综合征的猪群中被发现。随后在其他省份以及韩国也检测到了该病毒。鉴于该病毒引发疫情的可能性，需要快速、灵敏且特异的PCV4检测方法，同时也需要通过阐明PCV4的全基因组来推动进一步的流行病学研究。本研究旨在实现这两个目标。
对来自四川省猪场的55份血液样本、2份猪组织样本、9份唾液拭子和1份精液样本进行了分析。基于中国参考毒株人工合成了1770 bp的病毒基因组，并设计了该基因的引物和探针。然后，将扩增的目标片段克隆到pMD19-T载体中，并使用一系列稀释的重组质粒生成标准曲线。建立了优化的实时荧光定量TaqMan PCR方法。
本研究结果表明，所建立的方法对PCV4具有特异性，对其他病毒无特异性，扩增效率为99.6%，回归平方值（R）为1.000，检测限为2.2×10 DNA拷贝。该方法具有分析特异性和灵敏性，批内和批间变异系数低（<1.67%）。使用所建立的方法检测的67份临床样本中，有3份呈阳性（4%）。
本研究证实了PCV4在四川的存在，并为PCV4的快速检测提供了一种有前景的替代工具。
