Department of Biotechnology, Faculty of Science, Mahidol University, Rajadhevee, Rama VI Road, Bangkok 10400, Thailand.
J Virol Methods. 2013 Mar;188(1-2):51-6. doi: 10.1016/j.jviromet.2012.11.041. Epub 2012 Dec 7.
Yellow head virus (YHV) is a highly virulent pathogen that has caused severe mortality in cultivated shrimp (Penaeus monodon and Penaeus vannamei) in Thailand. There are several technologies that are applied to detect YHV for further control of the disease. RT-PCR is currently widely used in the laboratory, but it has some disadvantages related to cost, time-consuming and complexity. An alternative assay combines RT with loop-mediated isothermal amplification (LAMP) that not only provides high specificity, sensitivity and rapidity, but is also cheaper and more suitable for field applications in shrimp aquaculture than the RT-PCR. RT-LAMP is performed under isothermal conditions with a set of four to six primers designed to recognize six to eight distinct target sequences, and it has been combined with a chromatographic lateral-flow dipstick (LFD) to detect LAMP amplified product, which avoids the use of gel electrophoresis. In this study, RT-LAMP for the detection of YHV was developed by isothermal amplification at 65 °C for 45 min, followed by hybridization with an FITC-labeled DNA probe for 5 min and detected by LFD within 5 min (time required approximately 55 min, excluding RNA extraction and preparation time). The detection limit of RT-LAMP-LFD was 0.1 pg RNA extracted from shrimp infected with YHV equivalent to the nested RT-PCR, and no cross reaction was observed with other common shrimp viral pathogens. The LAMP method described in this study showed a rapid, high sensitivity and specificity and it is recommended as user-friendly for diagnosis of YHV in the field.
黄头病毒（YHV）是一种高致病性病原体，已导致泰国养殖虾（斑节对虾和凡纳滨对虾）大量死亡。有几种技术可用于检测 YHV，以进一步控制疾病。实时聚合酶链反应（RT-PCR）目前在实验室中广泛应用，但它存在一些与成本、耗时和复杂性相关的缺点。一种替代检测方法是将 RT 与环介导等温扩增（LAMP）相结合，该方法不仅具有高度特异性、敏感性和快速性，而且比 RT-PCR 更便宜，更适合虾类养殖中的现场应用。RT-LAMP 在等温条件下进行，使用一组四到六个引物来识别六个到八个不同的靶序列，并且已经与色谱横向流动条（LFD）结合使用来检测 LAMP 扩增产物，从而避免了使用凝胶电泳。在这项研究中，通过在 65°C 下等温扩增 45 分钟开发了用于检测 YHV 的 RT-LAMP，然后用 FITC 标记的 DNA 探针杂交 5 分钟，并在 5 分钟内通过 LFD 检测（大约需要 55 分钟，不包括 RNA 提取和准备时间）。RT-LAMP-LFD 的检测限为 0.1pg 从感染 YHV 的虾中提取的 RNA，与其他常见虾类病毒病原体无交叉反应。本研究中描述的 LAMP 方法显示出快速、高灵敏度和特异性，建议在现场用于 YHV 的诊断。
