鲤疱疹病毒Ⅱ型及Ⅲ型双重荧光定量PCR检测方法的建立与应用OA

To address the current challenges of low efficiency in single detection and difficulty in identifying co-infections,this study establishes a duplex fluorescence real-time PCR method for simultaneous and rapid detection of Cyprinid herpesvirus type Ⅱ(Cyprinid herpesvirus 2,CyHV-2)and type Ⅲ(Cyprinid herpesvirus 3,CyHV-3).This method seeks to address the current challenges associated with low efficiency in single-virus detection and the difficulty in distinguishing mixed infections.Utilizing the conserved regions of the ORF71 gene of CyHV-2 and the ORF140 gene of CyHV-3,three pairs of specific primers were designed and evaluated.Through the optimization of primer design,reaction systems,and reaction conditions,the specificity,sensitivity,and repeatability of the method were validated,culminating in the establishment of a duplex fluorescence qPCR method for the concurrent detection of CyHV-2 and CyHV-3.The study successfully established a duplex fluorescence qPCR detection method.The optimal primer sets were determined to be CyHV-2-F1&R1(Tm=82.78℃)and CyHV-3-F1&R1(Tm=89.22℃),with a clear separation of the two peaks observed in the melting curve.This study introduces a novel duplex fluorescence quantitative PCR(qPCR)method for the concurrent quantitative detection of CyHV-2 and CyHV-3.The method demonstrated a sensitivity of 14.8 copies/μL for CyHV-2 and 21.9 copies/μL for CyHV-3,with no cross-reactivity observed with grass carp reovirus type Ⅰ,Ⅱ,and Ⅲ(GCRV-Ⅰ,GCRV-Ⅱ,GCRV-Ⅲ)or Spring viraemia of carp virus(SVCV).The coefficients of variation for the cycle threshold(Ct)values and melting temperature(Tm)values were less than 4.0％and 0.2％,respectively.Clinical validation indicated a positive detection rate of 22.5％for CyHV-2 and 25.0％for CyHV-3,which is consistent with the results obtained by conventional PCR methods according to national and industry standards.This indicates that the dual fluorescent quantitative PCR method established in this study exhibits high sensitivity,specificity,and repeatability.In conclusion,this study is to establish a duplex fluorescence qPCR method for the simultaneous quantitative detection of CyHV-2 and CyHV-3.The method offers significant advantages,including rapid processing(completion within 2 hours),precise dual-target discrimination,high sensitivity(detection limit<30 copies/μL),and excellent repeatability.This method can be used for the rapid diagnosis and disease monitoring of CyHV-2 and CyHV-3 in aquaculture,serving as a reliable tool for the rapid differential diagnosis,prevention,and control of cyprinid herpesviruses,as well as a valuable reference for developing multiplex detection assays for other aquatic viruses.