对虾产品疫病双重荧光rpa方法的建立和初步应用.docxVIP

对虾产品疫病双重荧光RPA方法的建立和初步应用 张娜，谢艳辉，仇保封2,郑舒尹，斯泽恩，李家侨* (1.湛江海关技术中心，湛江524000； 2.南通海关，南通524000) 摘要：为实现对虾产品中两种病原体的简便、快速、高效的检测，本研究针对对虾传染 性皮下及造血器官坏死病毒和肝肠胞虫进行双重荧光RPA检测方法的研究。根据IHHNV 病毒和EHP的保守序列设计引物探针，建立双重荧光RPA方法，同时对方法的特异性、敏 感性和重复性进行验证，结果表明所建立的方法只对IHHNV和EHP有明显的扩增曲线； 用IHHNV和EHP阳性质粒进行倍比稀释检测，结果表明方法对IHHNV和EHP质粒检测 灵敏度可以达到1 xlO1 copies/^L；用10个重复质粒样品进行检测，结果证明方法重复性较 好。使用方法对进口的冷冻对虾样品检测，检测出1例EHP阳性和1例IHHNV阳性，同 时样品用荧光PCR方法进行比对检测，两种方法检测结果一致。本研究所建立的方法对于 两种疫病的区分检测具有重要的临床意义，可应用于养殖场、对虾产品生产企业和口岸实验 室疫病监控的快速筛查方法。 关键词:冷冻对虾；对虾传染性皮下及造血器官坏死病毒;肝肠胞虫;双重荧光 关键词: 冷冻对虾；对虾传染性皮下及造血器官坏死病毒; 肝肠胞虫; 双重荧光RPA； 临床应用 中图分类号：S855.9 文章编号： DOI： Establishment and preliminary application of dual fluorescent RPA for detection of shrimp products Zhang Na1, Xie Yanhui1, Qiu Baofeng2, Zheng Shuyin1, Si Zeen1, Li Jiaqiao1^ (l.Zhanjiang Customs Technology Center, ZhanJiang 524000,China； 2. Nantong Customs, Nantong 226004,China) Abstract: In order to realize the simple, rapid and efficient detection of the two pathogens in shrimp products, the dual fluorescence RPA detection method was studied for Infectious hypodermal and hematopoietic necrosis virus and Enterocytozoon hepatopenaei. Primers and probes were designed according to the conserved sequences of IHHNV virus and EHP, and a dual fluorescent RPA method was specificity,sensitivity and repeatability of the method were verified. The results showed that the method only amplified IHHNV and EHP; The sensitivity of IHHNV and EHP plasmids were 1x10, copies/)iL;Ten repeated plasmid samples were used for testing, and the results proved that the method had good repeatability. Using this method to detect the imported frozen prawn samples, one case was EHP positive and one case was IHHNV positive. At the same time, the samples were detected by Real-time PCR. The detection 收稿日期： 基金项目：海关总署科技项目(2019HK038, 2019HK025),湛江海关科技项目(2018GDK35) 作者简介：张娜(1982-)，女，高级兽医师，硕士研究生，从事出入境动物疫病检疫工作，E-mail： 通信作者：李家侨(1987-),女，汉族，兽医师，硕士研究生，从事出入境动物疫病检疫工作，E-mail：. results of the two methods were consistent. The metho