State Key Laboratory for Molecular Biology of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, 130112, China.
State Key Laboratory for Molecular Biology of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, 130112, China.
Mol Cell Probes. 2019 Dec;48:101448. doi: 10.1016/j.mcp.2019.101448. Epub 2019 Sep 12.
Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/μl and 2.93 × 101 copies/μl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.
貂细小病毒属病毒感染包括水貂、貉、狐、臭鼬和小熊猫在内的食肉动物物种。貂细小病毒属感染是养殖水貂发病和死亡的主要原因。在此，我们使用基于保守 VP2 基因的三组引物和一条探针，开发了一种用于检测和定量貂属细小病毒属病毒的直接 TaqMan qPCR 检测方法。Aleutian 貂病病毒 (AMDV) 和貉和北极狐细小病毒属 (RFAV) 的检测限分别为 4.06×101 拷贝/μl 和 2.93×101 拷贝/μl。内和间试验变异性均小于 2%。在 74 个食肉动物样本中，直接 TaqMan qPCR 的细小病毒属病毒阳性率为 62.2%（46/74），而 SYBR Green I qPCR 的阳性率仅为 40.5%（30/74）。这一结果表明，直接 TaqMan qPCR 比 SYBR Green I qPCR 更敏感。此外，直接 TaqMan qPCR 是一种快速灵敏的微升级液体样本检测方法，因为该检测方法采用直接碱性裂解法获得病毒 DNA，从而省去了繁琐的 DNA 提取步骤。总体而言，直接 TaqMan qPCR 检测方法具有高特异性、灵敏度和重现性，表明它可用于在流行病学和发病机制研究中检测和定量各种貂属细小病毒属病毒。
Zotero 插件