| 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 |
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| Amplicon Rescue Multiplex PCR (Arm-PCR): a Novel Tool for Simultaneous Detection of Seven Types of Fish Viruses |
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WANG Shengqiang1,2,3, GENG Weiguang1,2, SHI Chengyin1,2,4, LI Jin1,2, SU Zidan1,2,3
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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
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| 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 |
