鳗鲡疱疹病毒基础RPA及RPA-LFD检测方法的比较  

Comparison of basic RPA and RPA-LFD assays for Anguillid herpesvirus

作　　者：林而舒 LIN Ershu(Freshwater Fisheries Research Institute of Fujian Province,Fuzhou 350025,China)

机构地区：[1]福建省淡水水产研究所,福建福州350025

出　　处：《渔业研究》2024年第4期367-374,共8页Journal of Fisheries Research

基　　金：福建省属公益类科研院所基本科研专项(2023R1013003);国家大宗淡水鱼产业技术体系项目(CARS-45-41);福建省属公益类科研院所基本科研专项(2022R1014005)。

摘　　要：【目的】鳗鲡疱疹病毒(Anguillid herpesvirus,AngHV)是鳗鲡“脱黏败血综合征”的主要病原,对鳗鲡养殖产业造成了严重的经济损失。为达到早期预警和防止病毒扩增蔓延的目的,建立一套AngHV快速检测技术对中国鳗鲡养殖疾病防控具有重要意义。【方法】本研究根据AngHV ORF55设计了扩增引物和探针,建立了基于重组酶聚合酶等温扩增技术(RPA)的基础RPA及RPA侧向流层析试纸条法(RPA-LFD)检测方法,探究了不同时间和温度对基础RPA和RPA-LFD扩增效果的影响,并比较了2种方法的灵敏度、特异性和临床应用效果。【结果】本研究建立的AngHV基础RPA及RPA-LFD快速检测方法扩增产物大小为394 bp;2种检测方法都仅需20~40 min即可完成检测过程;温度的变化对检测方法的影响不大,37~43℃范围内均能得到较好的扩增效果;2种检测方法均与鳗鲡圆环病毒(Eel circovirus)、锦鲤疱疹病毒(Koi herpesvirus)和鲤疱疹病毒(Cyprinid herpesvirus)无交叉反应,表现出良好的特异性;RPA-LFD检测方法具有更高的检测灵敏度,检出限至1.32×10^(0)copies/mL,基础RPA检出限至1.32×10^(3)copies/mL;检测实验室保存的20份鳗鲡病料DNA,2种方法检测的结果一致,阳性率均为30%,表明AngHV基础RPA和RPALFD检测方法均具有良好的临床应用潜力。【结论】AngHV基础RPA和RPA-LFD检测方法十分适用于基层科研单位、鳗鲡产品加工场及养殖场的病毒检测,丰富了AngHV检测手段的可选性。[Objective]Eel is a major freshwater aquaculture economic species in China.Eel is rich in nutrients and delicious meat,which is very popular among consumers.However,with the continuous development of intensive aquaculture mode,there are more and more kinds of diseases in eel culture,among which the danger of viral diseases is particularly serious.Anguillid herpesvirus(AngHV)poses a significant threat to eel(Anguilla)breeding,often resulting in economic losses.Developing rapid detection technologies for AngHV is crucial for disease prevention and control in China’s eel industry.[Methods]AngHV DNA was extracted from liver and gill tissues of diseased eels in the laboratory using a DNA extraction kit.This study designed amplification primers and probes targeting AngHV ORF55 to establish basic RPA and RPA coupled with lateral flow detection(RPA-LFD)methods.The effects of time and temperature variations on RPA and RPA-LFD amplification were investigated.The sensitivity,specificity,and clinical applicability of both methods were compared.[Results]Both basic RPA and RPA-LFD assays amplified products of 394 bp within 20 to 40 minutes.The basic RPA assay time exceeded 80 min,which easily led to the appearance of stray bands.When the RPA-LFD detection time was extended to 160 min,the negative control group was prone to false positives.Temperature variations between 37℃ to 43℃ minimally affected amplification efficiency.It indicated that the change in temperature has little effect on the amplification results.Specificity tests showed no cross-reactivity with Eel circovirus,Cyprinid herpesvirus,and Koi herpesvirus.RPA-LFD exhibited high sensitivity with a detection limit of 1.32×10^(0) copies/mL,while basic RPA had a limit of 1.32×10^(3) copies/mL.Testing 20 clinical eel samples yielded consistent results between the two assays,with a 30%positive detection rate,indicating promising clinical applicability for both methods.[Conclusion]In this study,the primers of basic RPA and RPA-LFD were designed in the conserved reg

关 键 词：鳗鲡 鳗鲡疱疹病毒(AngHV) 重组酶聚合酶等温扩增(RPA) 重组酶聚合酶等温扩增-侧向流层析试纸条法(RPA-LFD) 

分 类 号：S943[农业科学—水产养殖]