FTA卡保存对虾传染性皮下和造血组织坏死病毒DNA洗脱方法的优化  

Optimization of DNA Elution Method of Infectious Hypodermic and Hematopoietic Necrosis Virus Preserved by FTA Card

作　　者：连新宇 王秀华[2] 李晨[2] 张庆利[2] 苟紫玥 吕若萱 杨冰[2] LIAN Xinyu;WANG Xiuhua;LI Chen;ZHANG Qingli;GOU Ziyue;LÜRuoxuan;YANG Bing(College of Fisheries,Zhejiang Ocean University,Zhoushan 316022,China;Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Affairs,Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity,Qingdao 266071,China)

机构地区：[1]浙江海洋大学水产学院,浙江舟山316022 [2]中国水产科学研究院黄海水产研究所、青岛海洋科技中心海洋渔业科学与食物产出过程功能实验室、农业农村部海水养殖病害防治重点实验室、青岛市海水养殖流行病学与生物安保重点实验室,山东青岛266071

出　　处：《渔业科学进展》2024年第3期193-202,共10页Progress in Fishery Sciences

基　　金：中国水产科学研究院基本科研业务费(2022GH01);中国水产科学研究院黄海水产研究所级基本科研业务费(20603022021012);国家重点研发计划(2019YFD0900101);国家现代农业产业技术体系(CARS48)共同资助。

摘　　要：传染性皮下和造血组织坏死病毒(infectious hypodermal and haematopoietic necrosis virus,IHHNV)是危害虾类健康养殖的重要病原,为寻找一种快捷保存及分离IHHNV DNA的方法,为后续研究提供完整的核酸材料,选用FTA(flinders technology associates)卡为保存介质,以FTA纯化试剂、TE(Tris-EDTA)缓冲液及去离子水为基础洗脱液,设计7种FTA卡黏附DNA洗脱方法,通过荧光定量PCR检测方法检验不同核酸洗脱分离效果及最低的点膜核酸量。结果显示,于4 mm2的FTA卡上,点样体积为2.5μL,用洗脱液作为模板时,最低点膜核酸浓度需要1.47×104copies/μL以上,可获得最佳的检测灵敏度和100%检出率的洗膜方法为50μL TE缓冲液于95℃下浸洗5 min;用膜片做模板,点膜核酸浓度需要1.82×103copies/μL以上,室温(20~25℃)条件下,用FTA纯化试剂洗脱3次,再用TE缓冲液洗脱2次,洗脱时间均为5 min,可获得最佳的检测灵敏度和100%的准确度。以FTA卡保存对虾白斑综合征病毒(white spot syndrome virus,WSSV)、虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)、虾十足目虹彩病毒1(Decapod iridescent virus 1,DIV1)、偷死野田村病毒(covert mortality noda virus,CMNV)、致急性肝胰腺坏死副溶血弧菌(Vibrio parahaemolyticus,VpAHPND)核酸,测试所建洗脱方法的效果,证实了该方法对其他虾类病原核酸的洗脱具有通用性。该研究给出了FTA卡保存和洗脱IHHNV DNA的适用性方案,为野外对虾样品采集、病毒核酸样品跨区域传递的保存和运输条件提供了科学数据。Flinders technology associates(FTA)card(Whatman®)is a paper-based matrix designed to fix,purify,and store genetic material from various biological sources.It can conveniently and quickly preserve nucleic acids and may fulfil the requirements of long-distance and cross-border sample transportation.The FTA card can store and transport tissue,nucleic acid,and other sample types at room temperature(20–25℃).Nucleic acid can be extracted directly for detection and be sent by express as an ordinary parcel without being treated as dangerous or as special goods,eliminating tedious processes,saving time,and ensuring sample quality.It is widely used in the human and animal medicine field.It has been successfully used for the storage and transportation of livestock pathogens and viral nucleic acids.In terms of aquatic animals,the FTA card has been used by researchers to store white spot syndrome virus(WSSV)and the shrimp Enterozoon hepatopoaei(EHP):However,there is no relevant research report on the elution effect of the nucleic acid stored in the FTA card,which affects the application of the FTA card.The infectious hypodermal and haematopoietic necrosis virus(IHHNV)is an important shrimp pathogen and severely impacts the shrimp culture industry.It was first found in Hawaii,United States,in 1981 and then spread to several countries,including to Australia,Singapore,Malaysia,South Korea,Brazil,and China.The IHHNV infecting Penaeus vannamei does not cause high mortality,but growth would become slow and deformed,resulting in great economic losses.Early detection and prevention management are particularly important in current situations,which lack effective control measures for the disease.Several IHHNV detection methods have been established that use molecular biological methods,including conventional polymerase chain reaction(PCR)and real-time PCR(these are recommended in the aquatic animal disease diagnosis Manual of the World Organization for Animal Health).Nucleic acid extraction by the above methods meets the requireme

关 键 词：IHHNV FTA卡 洗脱方法 优化 

分 类 号：S949[农业科学—水产养殖]