Aquatic Technology Laboratories, Agricultural Technology Research Institute, No. 1, Ln. 51, Dahu Rd., Xiangshan Dist, 300 Hsinchu, Taiwan.
Animal Technology Laboratories, Agricultural Technology Research Institute, Hsinchu, Taiwan.
J Virol Methods. 2017 Nov;249:94-101. doi: 10.1016/j.jviromet.2017.08.009. Epub 2017 Aug 20.
A loop-mediated isothermal amplification (LAMP) assay was used for rapid canine parvovirus (CPV) diagnosis. To reduce the time required and increase the sensitivity of the assay, an immunocapture (IC) technique was developed in this study to exclude the DNA extraction step in molecular diagnostic procedures for CPV. A polyclonal rabbit anti-CPV serum was produced against VP2-EpC that was cloned via DNA recombination. The polyclonal anti-VP2-EpC serum was used for virus capture to prepare microtubes. IC-LAMP was performed to amplify a specific CPV target gene sequence from the CPV viral particles that were captured on the microtubes, and the amplicons were analyzed using agarose electrophoresis or enzyme-linked immunosorbent assay (IC-LAMP-ELISA) and lateral-flow dipstick (IC-LAMP-LFD). The detection sensitivities of IC-LAMP, IC-LAMP-ELISA, and IC-LAMP-LFD were 10, 10, and 10 TCID/mL, respectively. Using the IC-LAMP-ELISA and IC-LAMP-LFD assays, the complete CPV diagnostic process can be achieved within 1.5h. Both of the developed IC-LAMP-based assays are simple, direct visual and efficient techniques that are applicable to the detection of CPV.
环介导等温扩增（LAMP）法用于快速诊断犬细小病毒（CPV）。为了减少所需时间并提高检测的灵敏度，本研究开发了一种免疫捕获（IC）技术，以排除 CPV 分子诊断程序中的 DNA 提取步骤。针对通过 DNA 重组克隆的 VP2-EpC 产生了多克隆兔抗 CPV 血清。使用多克隆抗 VP2-EpC 血清进行病毒捕获，以制备微管。IC-LAMP 用于从捕获在微管上的 CPV 病毒粒子扩增特定的 CPV 靶基因序列，并用琼脂糖电泳或酶联免疫吸附试验（IC-LAMP-ELISA）和侧向流动纸条（IC-LAMP-LFD）分析扩增子。IC-LAMP、IC-LAMP-ELISA 和 IC-LAMP-LFD 的检测灵敏度分别为 10、10 和 10 TCID/mL。使用 IC-LAMP-ELISA 和 IC-LAMP-LFD 检测，完整的 CPV 诊断过程可以在 1.5 小时内完成。两种基于 IC-LAMP 的检测方法都简单、直接、直观且高效，适用于 CPV 的检测。
