Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, Zhejiang, People's Republic of China.
Arch Virol. 2010 Mar;155(3):385-9. doi: 10.1007/s00705-010-0593-4. Epub 2010 Jan 27.
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. In this report, a 20-min LAMP amplification of the DPOL gene of infectious spleen and kidney necrosis virus (ISKNV) using a biotin-labeled primer was combined with lateral flow dipstick (LFD) chromatography for rapid and simple visual detection of ISKNV-specific amplicons. The LFD process involves a 5-min specific hybridization with an FITC-labeled DNA probe to confirm the presence of complement ISKNV amplicons that were biotinated in LAMP. The resulting DNA duplexes, consisting of labeled probes and amplicons, migrate along the LFD strip by chromatography for 5 min and are trapped at the test line and visualized by biotin labeling. The detection limit of ISKNV by LAMP-LFD was 10 copies. The results show that the LAMP-LFD method has the advantages of better sensitivity and speed and less dependence on equipment than the standard PCR for specifically detecting low levels of ISKNV DNA, and this can be useful in the field as a routine diagnostic tool.
环介导等温扩增（LAMP）允许在等温条件下快速扩增核酸。在本报告中，使用生物素标记的引物对传染性造血器官坏死病毒（ISKNV）的 DPOL 基因进行了 20 分钟的 LAMP 扩增，然后结合侧流层析（LFD）色谱法，快速简便地可视化检测 ISKNV 特异性扩增子。LFD 过程涉及与 FITC 标记的 DNA 探针特异性杂交 5 分钟，以确认存在互补的 ISKNV 扩增子，这些扩增子在 LAMP 中被生物素化。形成的 DNA 双链体，由标记的探针和扩增子组成，通过色谱法在 5 分钟内迁移，并在测试线上被捕获并通过生物素标记可视化。LAMP-LFD 对 ISKNV 的检测限为 10 拷贝。结果表明，与标准 PCR 相比，LAMP-LFD 方法具有更好的灵敏度和速度优势，并且对设备的依赖性更低，可特异性检测低水平的 ISKNV DNA，这在现场作为常规诊断工具可能很有用。
