Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick
Rapid and sensitive detection of white spot syndrome virus by loop-mediated isothermal amplification combined with a lateral flow dipstick
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DOI：
10.1016/j.mcp.2008.12.003
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发表时间：
2009-04-01
影响因子：
3.3
通讯作者：
Flegel, Timothy W.
中科院分区：
文献类型：
--
作者：
Jaroenrama, Wansadaj;Kiatpathomchai, Wansika;Flegel, Timothy W.
环介导等温扩增（LAMP）允许使用一组识别六种不同靶序列的四种特异性设计的引物在等温条件下快速扩增核酸。它可以与色谱侧流试纸条（LFD）结合，用于高度特异性，快速和简单的视觉检测WSSV特异性扩增子。使用该方案，30分钟扩增，然后与FITC标记的DNA探针杂交5分钟，5分钟LFD导致在LFD测试线处捕获的DNA扩增子的可视化。因此，10分钟的快速DNA提取，然后与LFD检测相结合的LAMP导致总检测时间约为50分钟。检测灵敏度与其他常用的方法套式PCR检测WSSV，但有额外的优势，减少检测时间，确认扩增子的身份杂交和消除电泳致癌溴化乙锭。(C)2008爱思唯尔有限公司保留所有权利。
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions using a set of four specifically designed primers that recognize six distinct target sequences. It can be combined with a chromatographic lateral flow dipstick (LFD) for highly specific, rapid and simple visual detection of WSSV-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 commonly-used methods for nested PCR detection of WSSV but had the additional advantages of reduced assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide. (C) 2008 Elsevier Ltd. All rights reserved.