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

摘要

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 ofTaq DNA polymerase, Mg2+, dNTP and Primer Mix shown as follows. Reaction mixture (50μl) consisted of 1.0μlTaq DNA polymerase (2.5 U/μl), 5μl 10×PCR Buffer (20 mmol/L Mg2+), 5μl dNTP (2.5 mmol/L each), 9μl 10×Primer Mix (2μmol/L), and 1μl 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), andmatrix 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.%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)1.0μl，10×PCR Buffer(含20 mmol/L的Mg2+)5μl，dNTP(各2.5 mmol/L)5μl，10×Primer Mix(各2μmol/L)9μl，模板1μl，ddH2O补足至50μl，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。