海洋渔业2025，Vol.47Issue(5)：619-628,10.
鲤疱疹病毒2型和3型双重TaqMan荧光定量PCR检测方法的建立与应用
Establishment and application of double TaqMan qPCR for detection of cyprinid herpesvirus 2 and cyprinid herpesvirus 3
摘要
Abstract
Cyprinid herpesvirus 2(CyHV-2),primarily impacts Carassius auratus var.and related species,while Cyprinid herpesvirus 3(CyHV-3)is responsible for Koi herpesvirus disease(KHVD),causing significant mortality in Cyprinus carpio,Cyprinus carpio haematopterus,and their hybrids.Both viruses are characterized by high pathogenicity and rapid transmission,leading to substantial economic losses worldwide.To address the critical diagnostic challenge posed by CyHV-2 and CyHV-3,this study focused on developing a highly efficient and precise detection method.Specifically,we engineered a novel dual TaqMan quantitative real-time PCR(qPCR)assay capable of concurrently identifying both pathogens within a single reaction.This multiplex approach was achieved by designing two unique pairs of sequence-specific primers alongside their corresponding dual-labeled hydrolysis probes(TaqMan probes),each set precisely targeted to amplify and detect highly conserved sequences located within the essential DNA polymerase genes of CyHV-2 and CyHV-3,respectively.Following rigorous optimization of reaction conditions,including annealing temperature,primer concentration,a multiplex qPCR protocol was established.And the specificity,sensitivity,and repeatability of the method were evaluated.Specificity evaluation confirmed that the diagnostic assay demonstrated no detectable cross-reactivity with a panel of other significant and prevalent aquatic viruses.Sensitivity analyses demonstrated a detection limit of 10 copies·μL-1 for both targets,indicating high analytical sensitivity.Reproducibility was evidenced by low intra-assay and inter-assay coefficients of variation(CV＜3.0％across triplicate runs).Clinical validation utilized 37 archived Carassius auratus var.samples and 78 Cyprinus carpio haematopterus tissues.Results showed 100％concordance with national(GB/T 36194-2018)and industry(SC/T 7212.1-2011)standard diagnostic methods.Additionally,co-infection testing of five mixed clinical samples correctly identified both viruses,confirming multiplexing capability.The established dual TaqMan qPCR assay offers high specificity,exceptional sensitivity,strong reproducibility,and excellent clinical sample compatibility.These attributes strongly support its utility in routine diagnostics,facilitating rapid and timely detection of CyHV-2 and CyHV-3 in various aquaculture settings,as well as in epidemiological surveillance to monitor viral spread patterns.Importantly,the assay also serves as a valuable tool for differential diagnosis,aiding in accurately distinguishing between the two herpesviruses that often co-circulate in aquatic environments.Overall,this duplex TaqMan qPCR method not only advances technical capabilities but also provides practical,actionable tools for clinical practice,contributing to better improved disease management and control of CyHV-2 and CyHV-3 in aquaculture product.关键词
鲤疱疹病毒2型/鲤疱疹病毒3型/双重TaqMan qPCRKey words
cyprinid herpesvirus 2/cyprinid herpesvirus 3/double TaqMan qPCR分类
农业科技引用本文复制引用
陈永聪,方勤美,柯翎,施少华..鲤疱疹病毒2型和3型双重TaqMan荧光定量PCR检测方法的建立与应用[J].海洋渔业,2025,47(5):619-628,10.基金项目
福建省科技计划公益类专项(2020R10270010、2021R1027008) （2020R10270010、2021R1027008）
