鳗鲡疱疹病毒实时荧光重组酶辅助扩增（RAA）检测方法的建立及应用

doi:10.16535/j.cnki.dlhyxb.2023-216

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大连海洋大学学报 ›› 2024, Vol. 39 ›› Issue (2) : 234-240. DOI: 10.16535/j.cnki.dlhyxb.2023-216

鳗鲡疱疹病毒实时荧光重组酶辅助扩增（RAA）检测方法的建立及应用

陈曦,杨金先,葛均青*

作者信息 +

Development and application of a real-time fluorescence recombinase- aided amplification method for detection of Anguillid herpesvirus

CHEN Xi，YANG Jinxian，GE Junqing*

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文章历史 +

摘要

为建立鳗鲡疱疹病毒(Anguillid herpesvirus，AngHV)的实时荧光重组酶辅助扩增(recombinase-aided amplification，RAA)检测方法，根据AngHV的ORF95基因序列设计引物和探针，优化反应温度，确定反应时间，并应用该方法对采集的临床样品进行检测。结果表明：本研究中建立的实时荧光RAA检测方法的最佳反应温度为39 ℃，反应时间为20 min；实时荧光RAA检测方法的灵敏度、特异性及重复性评价显示，RAA检测AngHV的最低检测量为1×102 copies/μL，可特异性地检测AngHV，与美洲鳗鲡腺瘤病毒(American eel adomavirus，AEAdoV)、蛙虹彩病毒(Rana grylio virus，RGV)、鲤疱疹病毒(Koi herpesvirus，KHV)和对虾白斑综合征病毒(White spot syndrome virus，WSSV)均无交叉反应；实时荧光RAA检测方法的组内和组间变异系数均小于5%，表明实时荧光RAA检测方法灵敏度高、特异性强和重复性好；应用该方法对采集的25份临床样品进行检测显示，实时荧光RAA检测方法与qPCR方法的检出率一致，均为92%，高于普通PCR的检出率(76%)。研究表明，本研究中所建立的鳗鲡疱疹病毒实时荧光重组酶辅助扩增RAA检测方法快速、灵敏、可靠、准确，可用于AngHV的临床快速检测和流行病学调查。

Abstract

In order to establish a real-time fluorescence recombinase-aided amplification (RAA) method for detection of Anguillid herpesvirus (AngHV), a primer pair and probe targeting ORF95 sequence of AngHV was designed, followed by optimizing the reaction temperature and fixing the reaction time. The results showed that the established real-time fluorescence RAA had optimal reaction temperature of 39 ℃, with the reaction time of 20 min. The further evaluation of sensitivity, specificity, and repeatability showed that the detection method had the minimum AngHV detection concentration of 1×102 copies/μL and had high specificity on AngHV, and showed no cross amplification reaction with American eel adomavirus(AEAdoV), Rana grylio virus(RGV), Koi herpesvirus(KHV), and White spot syndrome virus (WSSV). There was less than 5% of co-efficiency of variation within and between groups for the method which was then applied to analyse 25 collected samples. It was found that the detection rate of the real-time fluorescence RAA was consistent with that by qPCR of 92%, but much higer than PCR with the detection rate of 76%. These results revealed that the established RAA method for detection of AngHV was rapid, sensitive, reliable, and accurate, which is used for rapid clinical detection and conducting epidemiological investigations on AngHV.

导出引用

陈曦, 杨金先, 葛均青. 鳗鲡疱疹病毒实时荧光重组酶辅助扩增（RAA）检测方法的建立及应用[J]. 大连海洋大学学报, 2024, 39(2): 234-240 https://doi.org/10.16535/j.cnki.dlhyxb.2023-216

CHEN Xi, YANG Jinxian, GE Junqing. Development and application of a real-time fluorescence recombinase- aided amplification method for detection of Anguillid herpesvirus[J]. Journal of Dalian Fisheries University, 2024, 39(2): 234-240 https://doi.org/10.16535/j.cnki.dlhyxb.2023-216

中图分类号： S 941.41