2024 05 v.40 1033-1046
大口黑鲈弹状病毒qPCR检测方法的建立与临床应用
基金项目(Foundation):
浙江省农业重大技术协同项目（项目号：2022XTTGSC01）,题目：大口黑鲈养殖产业关键技术集成创新与示范;;
湖州市公益性应用研究项目（项目号：2021GZ24）,题目：大口黑鲈弹状病毒减毒活疫苗的研制及初步应用;;
浙江省科技厅院所专项（项目号：2024YSZX02）,题目：鳜鲈绿色养殖关键技术研究与示范~~
邮箱(Email):
sjinyu@126.com;panxiaoyi@163.com;
DOI:
10.13242/j.cnki.bingduxuebao.004564
中文作者单位:
浙江省淡水水产研究所农业农村部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室;上海海洋大学水产与生命学院;
摘要(Abstract):
大口黑鲈弹状病毒病是引起大口黑鲈幼苗暴发性死亡最严重的疾病，为满足大口黑鲈幼苗弹状病毒（Micropterus salmoides rhabdovirus, MSRV）的快速检测，建立大口黑鲈弹状病毒SYBR Green qPCR检测方法。根据大口黑鲈弹状病毒核蛋白基因的保守序列设计qPCR引物，对反应体系进行优化，制作标准曲线，并进行灵敏度、重复性和特异性测试，建立染料法qPCR检测方法，并在临床标本和人工感染鱼组织样品中进行应用。结果表明，构建的标准曲线，模板量与Ct值之间具有良好的线性关系，相关系数R2为0.9994，检测灵敏度达到4.5拷贝/μL，除MSRV、鳜弹状病毒（Sinipercachuatsirhabdovirus,SCRV）、杂交鳢弹状病毒（Hybridsnakeheadrhabdovirus,HSHRV）和斑鳢弹状病毒（Channamaculatarhabdovirus,CMRV）外，对其他7种病毒大口黑鲈蛙虹彩病毒（Largemouth bass ranavirus, LMBV）、传染性脾肾坏死病毒（Infectious spleen and kidney necrosis virus, ISKNV）、神经坏死病毒（Nervous necrosis virus, NNV），传染性造血器官坏死病毒（Infectious hematopoietic necrosis virus,IHNV）、鲤春病毒血症病毒（Spring viraemia of carp virus, SVCV）、草鱼呼肠孤病毒（Grass carp reovirus, GCRV）和黄颡鱼小RNA病毒（Yellow catfish picornavirus, YCPrV）都无扩增荧光信号；在对45份临床样品的检测中，建立的qPCR检出阳性率比套式PCR高11.11%；对人工感染MSRV的不同规格（1 g/ind、3 g/ind、8 g/ind）大口黑鲈各组织检测结果显示，脾脏中病毒载量最高，并且大规格鱼苗（8 g/ind）组织中病毒载量要低于小规格鱼苗（1 g/ind、3 g/ind），表明小规格鱼苗更易感。以上结果表明，建立的大口黑鲈弹状病毒SYBR Green qPCR检测方法是一种适合临床样品检测的特异性强、灵敏度高、稳定性好的MSRV定量方法。该方法的建立为MSRV疾病检疫提供了可靠的技术手段。
关键词(KeyWords):
大口黑鲈;;弹状病毒;;qPCR;;病毒检测
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参考文献
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[23]Tong G, Wei X, Yin W, et al. Development of a liquid chip technique to simultaneously detect spring viremia of carp virus, infectious hematopoietic necrosis virus, and viral hemorrhagic septicemia of salmonids[J]. J AOAC Int, 2017, 100(1):159-164. DOI:10. 5740/jaoacint. 16-0066.
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[25]Dettinger L, Gigante C M, Strohecker M, et al.Potential early rabies infection detected in two raccoon cases by LN34 pan-lyssavirus real-time RT-PCR assay in Pennsylvania[R/OL]. bioRxiv, 2021. 429287.DOI:10. 1101/2021. 02. 01. 429287.
[26]Fu X, Lin Q, Liang H, et al. The biological features and genetic diversity of novel fish rhabdovirus isolates in China[J]. Arch Virol, 2017, 162(9):2829-2834.DOI:10. 1007/s00705-017-3416-z.
[27]Zeng W, Wang Q, Wang Y, et al. Genomic characterization and taxonomic position of a rhabdovirus from a hybrid snakehead[J]. Arch Virol, 2014, 159(9):2469-2473. DOI:10. 1007/s00705-014-2061-z.
[28]Kumar S, Stecher G, Li M, et al. MEGA X:Molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6):1547-1549. DOI:10. 1093/molbev/msy096.
[29]Wu Y J, Yi S F, Cheng Y, et al. A concise review on advancement of Micropterus salmoides rhabdovirus(MSRV):Current status and challenges[J]. Aquac Rep, 2023, 30:101551. DOI:10. 1016/j.aqrep. 2023. 101551.
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[38]梁红茹，蔡秀珠，范芷仪，等.鳜弹状病毒TaqMan荧光定量PCR检测方法的建立及应用[J].中国预防兽医学报，2019, 41(9):929-934. DOI:10. 3969/j.issn. 1008-0589. 201901040.
[39]刘春，曾伟伟，王庆，等.杂交鳢（斑鳢♀×乌鳢♂）弹状病毒TaqMan实时荧光定量PCR检测方法的建立及应用[J].水产学报，2014, 38(1):136-142. DOI:10. 3724/SP. J. 1231. 2014. 48778.
[40]刘慧芳，张莉娟，林强，等.弱毒疫苗株MSRV-SS-7的安全性及有效性评价[C]//2018年中国水产学会学术年会论文摘要集.西安，2018:63.
[2]农业农村部渔业渔政管理局，全国水产技术推广总站，中国水产学会编制.中国渔业统计年鉴-2023[M].北京：中国农业出版社，2023:25.
[3] Yi S, Wu Y, Gu X, et al. Infection dynamic of Micropterus salmoides rhabdovirus and response analysis of largemouth bass after immersion infection[J]. Fish Shellfish Immunol, 2023, 139:108922. DOI:10. 1016/j. fsi. 2023. 108922.
[4] Zhao L, Zhong Y, Luo M, et al. Largemouth bass ranavirus:Current status and research progression[J].Aquac Rep, 2023, 32:101706. DOI:10. 1016/j.aqrep. 2023. 101706.
[5] Xu Z, Liao J, Zhang D, et al. Isolation,characterization, and transcriptome analysis of an ISKNV-like virus from largemouth bass[J]. Viruses,2023, 15(2):398. DOI:10. 3390/v15020398.
[6] Cai J, Yu D, Xia H, et al. Identification and characterization of a nervous necrosis virus isolated from largemouth bass(Micropterus salmoides)[J]. J Fish Dis, 2022, 45(4):607-611. DOI:10. 1111/jfd. 13576.
[7] Liu Z G, Dong J J, Ke X L, et al. Isolation,identification, and pathogenic characteristics of Nocardia seriolae in largemouth bass Micropterus salmoides[J]. Dis Aquat Organ, 2022, 149:33-45.DOI:10. 3354/dao03659.
[8] Gao E B, Chen G. Micropterus salmoides rhabdovirus(MSRV)infection induced apoptosis and activated interferon signaling pathway in largemouth bass skin cells[J]. Fish Shellfish Immunol, 2018, 76:161-166.DOI:10. 1016/j. fsi. 2018. 03. 008.
[9]霍明赫，许炜迪，冯华超，等.狂犬病疫苗接种者血清对狂犬病病毒中和能力研究[J].病毒学报，2022, 38(2):378-384. DOI:10. 13242/j. cnki.bingduxuebao. 003949.
[10]Godahewa G I, Lee S, Kim J, et al. Analysis of complete genome and pathogenicity studies of the spring viremia of carp virus isolated from common carp(Cyprinus carpio carpio)and largemouth bass(Micropterus salmoides):an indication of SVC disease threat in Korea[J]. Virus Res, 2018, 255:105-116.DOI:10. 1016/j. virusres. 2018. 06. 015.
[11]Black E M, Lowings J P, Smith J, et al. A rapid RTPCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan?technology[J]. J Virol Meth, 2002, 105(1):25-35.DOI:10. 1016/S0166-0934(02)00062-9.
[12]Liu X, Zhang X, Xu Z, et al. Isolation, genomic and biological characterizations of a rhabdovirus from mandarin fish(Siniperca chuatsi)[J]. Aquaculture,2023, 563:738894.DOI:10. 1016/j.aquaculture. 2022. 738894.
[13]Lyu S J, Yuan X M, Zhang H Q, et al. Isolation and characterization of a novel strain(YH01)of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system[J]. J Zhejiang Univ Sci B, 2019, 20(9):728-739. DOI:10. 1631/jzus. b1900027.
[14]He R, Liang Q, Zhu N, et al. Earlier activation of interferon and pro-inflammatory response is beneficial to largemouth bass(Micropterus salmoides)against rhabdovirus infection[J]. Fishes, 2022, 7(2):90.DOI:10. 3390/fishes7020090.
[15]Qin Y, Zhang P, Zhang M, et al. Isolation and identification of a new strain Micropterus salmoides rhabdovirus(MSRV)from largemouth bass Micropterus salmoides in China[J]. Aquaculture, 2023, 572:739538. DOI:10. 1016/j. aquaculture. 2023. 739538.
[16]Li B Y, Qin J C, Shen Y F, et al. A therapeutic agent of ursolic acid demonstrates potential application in aquaculture[J]. Virus Res, 2023, 323:198965. DOI:10. 1016/j. virusres. 2022. 198965.
[17]Hu Y, Zhou Y, Shan L, et al. Combination therapy with Polygala tenuifolia Willd and Phellodendron chinense Schneid extracts provides effective protection against Micropterus salmoides rhabdovirus in largemouth bass[J]. Aquaculture, 2024, 579:740246. DOI:10. 1016/j. aquaculture. 2023. 740246.
[18]张敏琳，黄枫淇，左小玲，等.基于CRISPR/Cas13a系统建立大口黑鲈弹状病毒检测方法[J].水生生物学报，2024, 48(2):283-291.
[19]Ma D, Deng G, Bai J, et al. A strain of Siniperca chuatsi rhabdovirus causes high mortality among cultured largemouth bass in south China[J]. J Aquat Anim Health, 2013, 25(3):197-204. DOI:10. 1080/08997659. 2013. 799613.
[20]黄俊，朱凝瑜，张徐俞，等.一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法：CN114592091A[P].2022-06-07.
[21]Bustin S A, Benes V, Garson J A, et al. The MIQE guidelines:minimum information for publication of quantitative real-time PCR experiments[J]. Clin Chem,2009, 55(4):611-622. DOI:10. 1373/clinchem. 2008. 112797.
[22]Luan H, Wang Y, Li Y, et al. Development of a realtime quantitative RT-PCR to detect REV contamination in live vaccine[J]. Poult Sci, 2016, 95(9):2023-2029.DOI:10. 3382/ps/pew147.
[23]Tong G, Wei X, Yin W, et al. Development of a liquid chip technique to simultaneously detect spring viremia of carp virus, infectious hematopoietic necrosis virus, and viral hemorrhagic septicemia of salmonids[J]. J AOAC Int, 2017, 100(1):159-164. DOI:10. 5740/jaoacint. 16-0066.
[24]Lin Q, Zhao Y, Fu X, et al. Development of strandspecific real-time RT-PCR for the analysis of SCRV transcription and replication dynamics[J]. Microb Pathog, 2019, 129:146-151. DOI:10. 1016/j.micpath. 2019. 02. 001.
[25]Dettinger L, Gigante C M, Strohecker M, et al.Potential early rabies infection detected in two raccoon cases by LN34 pan-lyssavirus real-time RT-PCR assay in Pennsylvania[R/OL]. bioRxiv, 2021. 429287.DOI:10. 1101/2021. 02. 01. 429287.
[26]Fu X, Lin Q, Liang H, et al. The biological features and genetic diversity of novel fish rhabdovirus isolates in China[J]. Arch Virol, 2017, 162(9):2829-2834.DOI:10. 1007/s00705-017-3416-z.
[27]Zeng W, Wang Q, Wang Y, et al. Genomic characterization and taxonomic position of a rhabdovirus from a hybrid snakehead[J]. Arch Virol, 2014, 159(9):2469-2473. DOI:10. 1007/s00705-014-2061-z.
[28]Kumar S, Stecher G, Li M, et al. MEGA X:Molecular evolutionary genetics analysis across computing platforms[J]. Mol Biol Evol, 2018, 35(6):1547-1549. DOI:10. 1093/molbev/msy096.
[29]Wu Y J, Yi S F, Cheng Y, et al. A concise review on advancement of Micropterus salmoides rhabdovirus(MSRV):Current status and challenges[J]. Aquac Rep, 2023, 30:101551. DOI:10. 1016/j.aqrep. 2023. 101551.
[30]夏焱春，曹铮，蔺凌云，等.大口黑鲈主要病害研究进展[J].中国动物检疫，2018, 35(9):72-76. DOI:10. 3969/j. issn. 1005-944X. 2018. 09. 019.
[31]K?drak-Jab?ońska A, Budniak S, Krupa M, et al.Detection of Listeria spp. and Listeria monocytogenes in biological samples by SYBR Green I and TaqMan probebased real-time PCRs[J]. J Vet Res, 2017, 61(4):427-432. DOI:10. 1515/jvetres-2017-0069.
[32]Wang Y, Sun J, Guo X, et al. A SYBR Green I-based real-time polymerase chain reaction assay for detection and quantification of canine bufavirus[J]. Mol Cell Probes, 2021, 59:101762. DOI:10. 1016/j.mcp. 2021. 101762.
[33]Nagaraj T, Vasanth J P, Desai A, et al. Ante mortem diagnosis of human rabies using saliva samples:Comparison of real time and conventional RT-PCR techniques[J]. J Clin Virol, 2006, 36(1):17-23.DOI:10. 1016/j. jcv. 2006. 01. 009.
[34]张莉娟，杨圆圆，方伟，等.弹状病毒基因组及功能基因研究进展[J].基因组学与应用生物学，2018, 37(2):815-826. DOI:10. 13417/j. gab. 037. 000815.
[35]岳志芹，刘荭，梁成珠，等.实时定量RT-PCR检测鱼类传染性造血器官坏死病毒方法的建立与应用[J].水生生物学报，2008, 32(1):91-95. DOI:10. 3321/j.issn:1000-3207. 2008. 01. 015.
[36]Walker P J, BigarréL, Kurath G, et al. Revised taxonomy of rhabdoviruses infecting fish and marine mammals[J]. Animals(Basel), 2022, 12(11):1363.DOI:10. 3390/ani12111363.
[37]Zhu N, He R X, Liang Q, et al. Establishment and application of a SYBR Green I real-time quantitative PCR for detection of Micropterus salmoides rhabdovirus[J]. Pak J Zool, 2023, 1-6. DOI:10. 17582/journal.pjz/20221026041025.
[38]梁红茹，蔡秀珠，范芷仪，等.鳜弹状病毒TaqMan荧光定量PCR检测方法的建立及应用[J].中国预防兽医学报，2019, 41(9):929-934. DOI:10. 3969/j.issn. 1008-0589. 201901040.
[39]刘春，曾伟伟，王庆，等.杂交鳢（斑鳢♀×乌鳢♂）弹状病毒TaqMan实时荧光定量PCR检测方法的建立及应用[J].水产学报，2014, 38(1):136-142. DOI:10. 3724/SP. J. 1231. 2014. 48778.
[40]刘慧芳，张莉娟，林强，等.弱毒疫苗株MSRV-SS-7的安全性及有效性评价[C]//2018年中国水产学会学术年会论文摘要集.西安，2018:63.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004564
中图分类号:S943
引用信息:
[1]陈雪兰,蔺凌云,杨娜等.大口黑鲈弹状病毒qPCR检测方法的建立与临床应用[J].病毒学报,2024,40(05):1033-1046.DOI:10.13242/j.cnki.bingduxuebao.004564.
基金信息:
浙江省农业重大技术协同项目（项目号：2022XTTGSC01）,题目：大口黑鲈养殖产业关键技术集成创新与示范;; 湖州市公益性应用研究项目（项目号：2021GZ24）,题目：大口黑鲈弹状病毒减毒活疫苗的研制及初步应用;; 浙江省科技厅院所专项（项目号：2024YSZX02）,题目：鳜鲈绿色养殖关键技术研究与示范~~
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