Tu Po-An, Shiu Jia-Shian, Lee Shu-Hwae, Pang Victor Fei, Wang De-Chi, Wang Pei-Hwa
Henchung Branch, Livestock Research Institute, Council of Agriculture, Executive Yuan, No.1, Muchang Rd., Hengchun Township, Pingtung County 946, Taiwan; Hsinchu Branch, Livestock Research Institute, Council of Agriculture, Executive Yuan, No. 207-5, Bi-tou-mian, Wu-hoo village, Si-hoo Township, Miao-li County 36848, Taiwan; Department of Animal Science and Technology, National Taiwan University, No. 50, Ln. 155, Sec. 3, Keelung Rd., Taipei City 10672, Taiwan.
Department of Animal Science and Technology, National Taiwan University, No. 50, Ln. 155, Sec. 3, Keelung Rd., Taipei City 10672, Taiwan; Agriculture Experiment Station, College of Bioresources and Agriculture, National Taiwan University No. 50, Ln. 155, Sec. 3, Keelung Rd., Taipei City 10672, Taiwan.
J Virol Methods. 2017 May;243:98-104. doi: 10.1016/j.jviromet.2017.01.023. Epub 2017 Jan 31.
Caprine arthritis-encephalitis (CAE) in goats is a complex disease syndrome caused by a lentivirus. This persistent viral infection results in arthritis in adult goats and encephalitis in lambs. The prognosis for the encephalitic form is normally poor, and this form of the disease has caused substantial economic losses for goat farmers. Hence, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed in the present study for detecting the proviral DNA of caprine arthritis-encephalitis virus (CAEV). Under the optimal incubation conditions, specifically, 30min at 37°C for RPA followed by 5min at room temperature for LFD, the assay was found to be sensitive to a lower limit of 80pg of total DNA and 10 copies of plasmid DNA. Furthermore, there was no cross-reaction with other tested viruses, including goat pox virus and bovine leukemia virus. Given its simplicity and portability, this RPA-LFD protocol can serve as an alternative tool to ELISA for the primary screening of CAEV, one that is suitable for both laboratory and field application. When the RPA-LFD was applied in parallel with serological ELISA for the detection of CAEV in field samples, the RPA-LFD assay exhibited a higher sensitivity than the traditional method, and 82% of the 200 samples collected in Taiwan were found to be positive. To our knowledge, this is the first report providing evidence to support the use of an RPA-LFD assay as a specific and sensitive platform for detecting CAEV proviral DNA in goats in a faster manner, one that is also applicable for on-site utilization at farms and that should be useful in both eradication programs and epidemiological studies.
山羊的山羊关节炎-脑炎（CAE）是一种由慢病毒引起的复杂疾病综合征。这种持续性病毒感染会导致成年山羊患关节炎，羔羊患脑炎。脑炎型的预后通常较差，这种疾病形式给山羊养殖户造成了巨大的经济损失。因此，本研究开发了一种基于重组酶聚合酶扩增（RPA）和侧向流动试纸条（LFD）的更高效检测平台，用于检测山羊关节炎-脑炎病毒（CAEV）的前病毒DNA。在最佳孵育条件下，具体而言，RPA在37°C孵育30分钟，随后LFD在室温下孵育5分钟，该检测方法对总DNA下限80pg和质粒DNA 10个拷贝敏感。此外，与其他测试病毒，包括山羊痘病毒和牛白血病病毒没有交叉反应。鉴于其简单性和便携性，这种RPA-LFD方案可作为ELISA的替代工具，用于CAEV的初步筛查，适用于实验室和现场应用。当将RPA-LFD与血清学ELISA并行应用于检测现场样本中的CAEV时，RPA-LFD检测方法表现出比传统方法更高的灵敏度，在台湾收集的200个样本中有82%被发现呈阳性。据我们所知，这是第一份提供证据支持使用RPA-LFD检测方法作为一种特异性和灵敏的平台，以更快的方式检测山羊中CAEV前病毒DNA的报告，该方法也适用于农场现场使用，并且在根除计划和流行病学研究中都应该有用。
