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同步检测7种鱼类病毒的扩增子拯救多重PCR(Arm-PCR)方法的建立和应用
王胜强,耿伟光,史成银,李 晋,粟子丹
1.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.上海海洋大学水产与生命学院 上海 201306;3.青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071
摘要:
淋巴囊肿病毒(LCDV)、肿大细胞病毒属虹彩病毒(Mega)、赤点石斑鱼神经坏死病毒(RGNNV)、传染性造血器官坏死病毒(IHNV)、传染性胰脏坏死病毒(IPNV)、病毒性出血败血症病毒(VHSV)和传染性鲑鱼贫血症病毒(ISAV)是养殖鱼类主要的病毒性病原,危害巨大。为实现这7种病原的高通量、同步检测,本研究在分析这7种病毒基因序列的基础上,设计了9组扩增子拯救多重PCR(Arm-PCR)引物,并对扩增体系中的Taq酶、Mg2+、dNTP、Primer Mix浓度及退火温度等参数进行调整和优化,结合基因芯片检测技术,建立了同步检测7种鱼类病毒的Arm-PCR方法。优化后的Arm-PCR方法第一步PCR体系为:Taq酶(2.5 U/μl) 1.0 μl,10×PCR Buffer(含20 mmol/L的Mg2+) 5 μl,dNTP(各2.5 mmol/L) 5 μl,10×Primer Mix(各2 μmol/L) 9 μl,模板1 μl,ddH2O补足至50 μl,退火温度为56℃。研究结果显示,该方法可以在1支反应管内对上述7种病毒的9个致病基因同步进行扩增和检测,检测灵敏度分别为101 copies/μl (RGNNV、VHSV、ISAV-NS、ISAV-MA)、102 copies/μl (LCDV、Mega、IHNV、IPNV)和103 copies/μl (大菱鲆红体病虹彩病毒,TRBIV)。该方法特异性强,与半滑舌鳎、石斑鱼、大菱鲆和牙鲆基因组DNA不产生交叉反应。本研究建立的可同步检测7种鱼类病毒的Arm-PCR方法具有高通量、高灵敏度、高准确性的优势,能有效提高工作效率,在鱼类病毒的筛查和流行病学调查领域有广泛的应用前景。
关键词:  鱼类病毒  多重检测  高通量  多重PCR
DOI:10.11758/yykxjz.20150606001
分类号:
基金项目:国家科技支撑计划课题(2012BAD17B01)资助
Amplicon Rescue Multiplex PCR (Arm-PCR): a Novel Tool for Simultaneous Detection of Seven Types of Fish Viruses
WANG Shengqiang1,2,3, GENG Weiguang1,2, SHI Chengyin1,2,4, LI Jin1,2, SU Zidan1,2,3
1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306;4.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071
Abstract:
Major fish viruses that are severely harmful in aquaculture industry include Lymphocystis disease virus (LCDV), Megalocytivirus (Mega), red-spotted grouper nervous necrosis virus (RGNNV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV) and infectious salmon anaemia virus (ISAV). Here we developed a specific amplicon rescue multiplex PCR (Arm-PCR) combined with gene microarray technique for the simultaneous detection of the seven types of fish viruses. First we optimized the conditions of Arm-PCR such as the annealing temperature and the concentrations of Taq DNA polymerase, Mg2+, dNTP and Primer Mix shown as follows. Reaction mixture (50 μl) consisted of 1.0 μl Taq DNA polymerase (2.5 U/μl), 5 μl 10×PCR Buffer (20 mmol/L Mg2+), 5 μl dNTP (2.5 mmol/L each), 9 μl 10×Primer Mix (2 μmol/L), and 1 μl template. The annealing temperature was 56℃. This method could simultaneously produce specific amplicons in one tube. The detection sensitivity of the Arm-PCR was 101 copies/μl for RGNNV, VHSV, non-structural protein of ISAV (ISAV-NS), and matrix protein of ISAV(ISAV-MA), 102 copies/μl for LCDV, Mega, IHNV, and IPNV, and 103 copies/μl for TRBIV (Turbot reddish body iridovirus). The Arm-PCR did not cause cross reactions with genomic DNA from healthy fish such as half smooth tongue sole, grouper, turbot and flounder.
Key words:  Fish virus  Multiple detection  High throughput  Multiplex PCR