National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, Thailand.
J Virol Methods. 2011 Jan;171(1):21-5. doi: 10.1016/j.jviromet.2010.09.022. Epub 2010 Sep 29.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is an important shrimp pathogen that causes mortality in Penaeus stylirostris and stunting (called runt deformity syndrome or RDS) in Penaeus vannamei. Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of IHHNV-specific amplicons. Using this protocol, a 30-min amplification followed by 5 min hybridization with an FITC-labeled DNA probe and 5 min LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, 10 min for rapid DNA extraction followed by LAMP combined with LFD detection resulted in a total assay time of approximately 50 min. Detection sensitivity was comparable to other methods used commonly for nested PCR detection of IHHNV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide.
传染性皮下及造血组织坏死病毒（IHHNV）是一种重要的虾类病原体，可引起日本对虾死亡，凡纳滨对虾生长迟缓（称为侏儒畸形综合征或 RDS）。环介导等温扩增（LAMP）可在等温条件下快速扩增核酸。它可以与层析侧向流试纸条（LFD）结合，用于高度特异性、快速和简单的 IHHNV 特异性扩增子的目视检测。使用该方案，在 30 分钟的扩增后，用 FITC 标记的 DNA 探针杂交 5 分钟，然后用 LFD 杂交 5 分钟，可将 DNA 扩增子固定在 LFD 测试线上进行可视化。因此，快速 DNA 提取后进行 LAMP 与 LFD 检测，总检测时间约为 50 分钟。检测灵敏度与常用于巢式 PCR 检测 IHHNV 的其他方法相当，但具有额外的优势，包括缩短了检测时间、通过杂交确认扩增子的身份以及消除了致癌溴化乙锭的电泳。
