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.
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
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