摘要
青蟹呼肠孤病毒(mud crab reovirus,MCRV)和青蟹双顺反子病毒-1(mud crab dicistrovirus-1,MCDV-1)是从近年发生大规模死亡的养殖拟穴青蟹体内分离的、对青蟹具有致病性的两种病毒。它们常常同时存在,给青蟹养殖业带来巨大经济损失。在疾病的防治中,快速高效的病毒检测方法对疾病诊断以及及时采取有效地防治措施具有重要的意义。本研究利用MCRV和MCDV-1基因保守区设计4对引物,建立了可同时检测这两种病毒的双重巢式PCR(duplex-nested PCR)检测方法。研究发现该方法对两种病毒的检测限都可以达到10个拷贝,并与鲍肌肉萎缩病毒(AbSV)、虾类传染性表皮与造血组织坏死症病毒(IHHNV)、大菱鲆红体病虹彩病毒(TRBIV)、对虾白斑综合症病毒(WSSV)、鱼类神经坏死病毒(NNV)和贝类急性病毒性坏死症病毒(AVNV)等水生动物常见病毒均无交叉反应,可以特异性地检测两种病毒。本方法具有高灵敏度、高特异性和简便快捷等特点,可作为一种快速诊断工具,检测拟穴青蟹中的MCRV和MCDV-1基因。
Recent outbreaks of ＂sleeping disease＂ have caused mass mortality in cultured mud crab and widespread economic loss in southeast China. Two novel pathogens （Mud Crab Reovirus, MCRV and Mud Crab Dicistrovirus, MCDV-1） have been isolated from crab exhibiting ＂sleeping symptoms＂. Thus, there is an urgent need for the accurate and early detection of this pathogen to facilitate disease control. We developed a duplex nested-PCR protocol for the detection of MCRV and MCDV-1 in Scylla paramamosain. Two pairs of primers targeting the conserved regions of the two viruses genes were designed using Primer Premier 5.0. The outer primers for MCRV were ReoF （5＇-GCAAATTGAACTACTACTACTTA-3＇） and ReoR （5＇-GATTCCTATTGTCAACTATCTCA-3＇）, and the inner primers were ReoNF （5＇-ACTCATAGAGCAGTCATGGG-3＇） and ReoNR （5＇-ATATCGTCAGAATGTC GTTC-3＇）. The outer primers for MCDV-1 were DicF （5＇-GCACTGGGTACTCTTCCTG-3＇） and DicR （5＇-AC ACCTACCAAAGCCCTAC-3＇）, and the inner primers were DicNF （5＇5＇-GGATACTATGGATGATGTTTC-3＇） and DicNR （5＇- ACAAAATACCAGATAAAGCAA-3＇）. The PCR products were differentiated by size. The first PCR was carried out using a PCR mix consisting of 1μL DNA, 1μL MCRV outer primer （ReoF/ReoR, 0.5μmol/L）, 0.4 pL MCDV-1 outer primer （DicF/DicR, 0.2 μmol/L）, and dH20 （to a final volume of 20 μL）. The thermal cycle was： 94℃ for 3 min, followed by 30 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min, followed by a final extension at 72℃ for 10 min. The nested PCR was carried out using the same mix with 0.2μL template DNA （the product of the first PCR）, 0.8μL MCRV inner primer （ReoNF/ReoNR, 0.4μmol/L）, and 0.8 μL MCDV-1 inner primer （DicNF/DicNR, 0.4 μmol/L）. The conditions were the same as in the first PCR, except the annealing temperature was 53℃. We evaluated the specificity and sensitivity of the duplex nested-PCR. The assay did not exhibit any cross-reactivity with abalone shriveling syndrome-associated vi
出处
《中国水产科学》
CAS
CSCD
北大核心
2013年第4期808-815,共8页
Journal of Fishery Sciences of China
基金
广东省科技计划项目(2011A020102002)
广东省海洋渔业科技推广专项(A201001H04)
广东省教育部产学研结合项目(2010B090400519)
中国水产科学研究院基本科研业务费资助(2012TS06)
