摘要
建立鲤春病毒血症病毒(SVCV)、鲤疱疹病毒3(CyHV-3)、鲤鱼浮肿病毒(CEV)和病毒性神经坏死病病毒(VNNV)等4种鱼类常见病毒的环介导等温扩增技术(LAMP)联合检测方法。基于4种鱼类病毒的基因保守区分别设计LAMP引物组,优化反应体系和条件,筛选测试出特异性和灵敏度最佳的引物组,建立4种鱼类常见病毒的联合检测LAMP方法,同时以市售的荧光定量检测试剂盒测试结果作为对照,评估建立的联合检测LAMP方法的准确性。结果显示,筛选出的SVCV、CyHV-3、CEV和VNNV的LAMP引物组特异性均良好,优化后的体系反应条件均为63℃,60 min,其中SVCV和CyHV-3均可检测到10 fg/μL,CEV和VNNV可检测到1 fg/μL。应用建立的4种病毒联合LAMP检测方法和市售的各荧光定量PCR检测试剂盒对150份收集的组织样本进行平行测试,联合LAMP检测方法的准确率为100%。建立的鱼类病毒LAMP联检方法能够准确、高效、快速地在鱼类组织样本中同时对SVCV、CyHV-3、CEV和VNNV进行检测,适合水产养殖现场检测,对病毒的监测和快速诊疗起到重要作用。
A combined detection method using loop-mediated isothermal amplification(LAMP)is established for four common fish viruses,including spring viremia of carp virus(SVCV),cyprinid herpesvirus 3(CyHV-3),carp edema virus(CEV),and viral nervous necrosis virus(VNNV).LAMP primer sets are designed based on the gene conserved regions of SVCV,CyHV-3,CEV and VNNV,and the reaction system and reaction conditions are optimized.Primers with the best specificity and sensitivity are screened and tested,and a LAMP method for the combined detection of 4 common fish viruses is established at the same time,the accuracy of the established LAMP method is evaluated by comparing the results of commercially available fluorescence quantitative detection kits.The results show that the specificity of the screened LAMP primer sets of SVCV,CyHV-3,CEV and VNNV is good,and the reaction conditions of the optimized system are consistent at 63℃for 60 min,in which SVCV and CyHV-3 can be detected at 10 fg/μL,CEV and VNNV can be detected at 1 fg/μL.The established combined LAMP assay for four viruses and the commercial quantitative PCR assay kits are applied to 150 collected tissue samples for parallel testing,and the accuracy of the combined LAMP assay is 100%.The established LAMP method for the combined detection of four common fish viruses can accurately,efficiently and rapidly detect SVCV,CyHV-3,CEV and VNNV in fish tissue samples at the same time.It is suitable for on-site detection in aquaculture,and plays an important role in virus monitoring and rapid diagnosis and treatment.
作者
时国强
张妍
李贺杰
焦义然
张璜
张婵
石磊
董振国
SHI Guoqiang;ZHANG Yan;LI Hejie;JIAO Yiran;ZHANG Huang;ZHANG Chan;SHI Lei;DONG Zhenguo(Hebei Sanshi Biotechnology Co.,Ltd.,Shijiazhuang Hebei 050035,China;College of Life Sciences,Hebei Normal University,Shijiazhuang Hebei 050024,China;College of Food and Biology,Hebei University of Science and Technology,Shijiazhuang Hebei 050018,China)
出处
《河北省科学院学报》
CAS
2023年第6期33-40,76,共9页
Journal of The Hebei Academy of Sciences
基金
河北省产业创新创业团队项目(205A7601D)。
关键词
SVCV
CyHV-3
CEV
VNNV
LAMP方法
Spring viremia of carp virus
Cyprinid herpesvirus-3
Carp edema virus
Viral nervous necrosis virus
Loop-mediated isothermal amplification method
