南方农业学报2025，Vol.56Issue(8)：2651-2661,11.DOI:10.3969/j.issn.2095-1191.2025.08.024
鲈鳜主要病毒性病原三重荧光定量PCR检测方法的建立及应用
Development and application of a triplex fluorescence quantita-tive PCR detection method for major viral pathogens in Micropterus salmoides and Siniperca chuatsi
摘要
Abstract
[Objective]This study aimed to develop a triplex fluorescence quantitative PCR method that was able to si-multaneously detect ranavirus(RanaV),infectious spleen and kidney necrosis virus(ISKNV)and Perciformes fish Rhab-doviridae(PFRV),with a goal of providing a convenient and efficient detection method for quarantine,monitoring,and clinical diagnosis of RanaV,ISKNV,and PFRV.[Method]Specific primers and TaqMan-MGB probes were designed based on the conserved sequences of the RanaV-MCP gene,ISKNV-MCP gene and PFRV-G gene.Target gene fragments were cloned into the pGM-T vector to construct recombinant plasmids of pGM-T-MCPRanaV,pGM-T-MCPISKNV,and pGM-T-GPFRV.pGM-T-MCPRanaVand pGM-T-MCPISKNV served as the standard DNA for RanaV and ISKNV,respectively,while pGM-T-GPFRV was digested with SalⅠ and then subjected to in vitro transcription to obtain the PFRV standard RNA.With the obtained standards as templates,reaction conditions were optimized and the standard curves were established to con-duct the sensitivity,specificity,reproducibility tests and experiments of clinical application.[Result]The optimized reac-tion system of the triplex fluorescence quantitative PCR 30.0 μL:2×Fast One Step Probe RT-qPCR Mix 15.0 μL,primer RanaV-qF/RanaV-qR(10 μmol/L)0.6 μL,primer ISKNV-qF/ISKNV-qR(10 μmol/L)0.6 μL,primer PFRV-qF/PFRV-qR(10 μmol/L)0.6 μL,probe RanaV-qP(5 μmol/L)0.9 μL,probe ISKNV-qP(5 μmol/L)0.9 μL,probe PFRV-qP(5 μmol/L)1.2 μL,nucleic acid templates 3.0 μL,sterile DEPC-treated water added to adjust the final volume to 30.0 μL.The triplex fluorescence quantitative PCR method demonstrated high amplification efficiency for RanaV,ISKNV and PFRV stan-dards(Eff.>95.0%),exhibited a strong linear relationship of standard curves(RSq≥0.999),enabling simultaneous quanti-tative analysis of RanaV,ISKNV and PFRV.This method showed high detection sensitivities for 10 copies/reaction for RanaV,ISKNV and PFRV standards and 10 copies/mg for tissue samples of Micropterus salmoides and Siniperca chuats.No cross reaction was found with common fish pathogens such as grass carp reovirus(GCRV),tilapia lake virus(TiLV),and spring viremia of carp virus(SVCV).The Ct variation coefficients of intra-group and inter-group for the de-tection of RanaV,ISKNV and PFRV were lower than 2.00%.The triplex fluorescence quantitative PCR method was ap-plied to detect samples collected from aquaculture farms(95 pieces of Micropterus salmoides and 68 pieces of Siniperca chuats),and the RanaV,ISKNV,and PFRV positive rates were 19.0%,14.7%,and 4.3%,respectively.[Conclusion]The triplex fluorescence quantitative PCR method based on probe TaqMan-MGB could simultaneously detect RanaV,ISKNV,PFRV.It features high sensitivity,strong specificity,accuracy,reliability,simplicity,and high efficiency,pro-viding a more convenient and effective tool for fry quarantine,epidemic monitoring,and clinical diagnosis of Microp-terus salmoides and Siniperca chuats.In aquaculture in Giangxi,RanaV,ISKNV,PFRV infections are observed,as Mi-cropterus salmoides infected by RanaV and Siniperca chuats by ISKNV,and cases of mixed infection and latent infection without clinical symptom are reported.关键词
大口黑鲈/鳜/虹彩病毒(RanaV)/传染性脾肾坏死病毒(ISKNV)/鲈形目鱼类弹状病毒(PFRV)/三重荧光定量PCRKey words
Micropterus salmoides/Siniperca chuats/ranavirus(RanaV)/infectious spleen and kidney necrosis vi-rus(ISKNV)/Perciformes fish Rhabdoviridae(PFRV)/triplex fluorescence quantitative PCR分类
农业科技引用本文复制引用
谭红连,韦剑成,李莉萍,童桂香,苏美珍,韦信贤,杨明伟,陈静..鲈鳜主要病毒性病原三重荧光定量PCR检测方法的建立及应用[J].南方农业学报,2025,56(8):2651-2661,11.基金项目
广西重点研发计划项目(桂科农AB2506910047) （桂科农AB2506910047）
国家现代农业产业技术体系广西特色淡水鱼创新团队项目(nycytxgxcxtd-2021-08-02) （nycytxgxcxtd-2021-08-02）
广西农业科技自筹经费项目(Z202299) （Z202299）
广西水产遗传育种与健康养殖重点实验室自主研究项目(2022-A-03-01) Guangxi Key Research and Development Plan Project(Guikenong AB2506910047) （2022-A-03-01）
Guangxi Special Freshwater Fish Innovation Team Project of China Agriculture Research System(nycytxgxcxtd-2021-08-02) （nycytxgxcxtd-2021-08-02）
Guangxi Agricultural Science and Technology Self-financing Project(Z202299) （Z202299）
Independent Research Project of Guangxi Key Laboratory of Aquatic Genetic Breeding and Healthy Aquaculture(2022-A-03-01) （2022-A-03-01）
