对虾肝肠胞虫LAMP-LFD检测方法的建立及应用Establishment and Application of LAMP-LFD Method for Detection of Enterocytozoon hepatopenaei
李文泽,侯晓勇,杨世平,黄郁葱,简纪常,蔡双虎
摘要(Abstract)：
【目的】采用环介导等温扩增（LAMP）技术联合横向流动试纸条（LFD）快速检测对虾肝肠胞虫（Enterocytozoon hepatopenaei, EHP），为对虾养殖场早期疫病监测提供技术支持。【方法】针对对虾肝肠胞虫18S rDNA保守基因序列设计4条特异性引物（上游内引物FIP由生物素标记）和1条异硫氰酸荧光素（FITC）标记的探针，使用引物进行有生物素标记的LAMP反应，产物与FITC标记的探针完成杂交，杂交产物在LFD上进行结果展示。并测试该方法的特异性、灵敏度和临床应用效果。【结果】LAMP最佳反应条件是62℃下运行15 min，加入探针杂交5 min后在LFD试纸条上进行显色反应，能特异直观检测到对虾肝肠胞虫。建立的LAMP-FLD法特异性强且灵敏度高，较普通聚合酶链式反应方法灵敏度高100倍。采用本研究建立的LAMP-LFD方法，对肝肠胞虫、对虾白斑综合征病毒（White spot syndrome virus）、十足目虹彩病毒1(Decapod iridescent virus 1)和副溶血弧菌（Vibrio parahaemolyticus）人工感染的凡纳滨对虾（Litopenaeus vannamei）肝胰腺组织和疑似感染对虾肝肠胞虫的凡纳滨对虾自然样本进行检测，结果显示该LAMP-LFD方法能100%准确检测出肝肠胞虫阳性样本。【结论】本研究建立的LAMP-LFD方法对对虾肝肠胞虫进行可视化检测，特异强、灵敏度高、操作简单、仪器设备要求及检测成本低和耗时短，适用于基层养殖场对虾肝肠胞虫感染早期的即时检测，具有较高推广价值。
关键词(KeyWords)： 对虾肝肠胞虫;环介导等温扩增技术;横向流动试纸条;凡纳滨对虾
基金项目(Foundation): 广东省现代农业产业技术体系创新团队建设项目（2024CXTD25）
作者(Author): 李文泽,侯晓勇,杨世平,黄郁葱,简纪常,蔡双虎
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