刘明珠,黄静,程远,韦云依,牟容丽,黄琳,竺利波,陆兰天,柯珂,陈嘉,余庆,李鹏飞

• 1:广西水产生物技术与现代生态养殖重点实验室
• 2:广西渔业重大疫病防控与高效健康养殖产业技术工程研究中心
• 3:广西科学院
• 4:中国-东盟现代渔业产业技术转移示范中心

摘要(Abstract)：

【目的】利用核酸适体LYGV1c构建可快速、准确识别石斑鱼虹彩病毒（Grouper iridovirus Guangxi strain,SGIV-Gx）感染的酶联吸附法（Aptamer LYGV1c-based enzyme-linked apta-sorbent assay,LYGV1c-ELASA），为提高水产疫病检测及防控效率提供理论支持。【方法】基于生物素标记的LYGV1c(Bio-LYGV1c)开发核酸适体酶联吸附法（LYGV1c-ELASA）检测SGIV-Gx，通过对Bio-LYGV1c特异性、灵敏性、稳定性等实验评估其检测性能。通过珍珠龙胆石斑鱼（Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂）SGIV-Gx感染试验进行活体验证，同时用荧光定量PCR(qPCR)测定衣壳蛋白基因（MCP）的表达，与LYGV1c-ELASA进行比较，验证该酶联吸附法检测的可信度。【结果】LYGV1c-ELASA的方法可特异性地识别SGIV的感染，Bio-LYGV1c识别SGIV感染的最适工作浓度为500 nmol/L，最适孵育时间为20 min，最适结合温度为4～28℃，LYGV1c-ELASA最低检测限为5×103 mL-1。活体验证结果表明，随着注射的SGIV-Gx浓度的升高，LYGV1c-ELASA检测出的石斑鱼体内病毒450 nm处的光密度（OD450）的值升高；qPCR结果表明，SGIV-Gx的MCP的表达量升高，与LYGV1c-ELASA检测结果相一致。【结论】建立的LYGV1c-ELASA技术不仅可用于体外检测，同时也适用于活体检测。LYGV1c-ELASA技术可实现对石斑鱼养殖过程中石斑鱼虹彩病毒病的快速诊断、实时监控。

关键词(KeyWords)： 石斑鱼虹彩病毒;检测;核酸适体;酶联吸附法

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（32373175）;; 广西自然科学基金（2023JJG130007,2022GXNSFBA035521,2022JJA130074）;; 国家重点研发计划（2022YFD2401200）;; 国家现代农业产业技术体系广西创新团队项目（nycytxgxcxtd-2021-08-02）

作者(Author): 刘明珠,黄静,程远,韦云依,牟容丽,黄琳,竺利波,陆兰天,柯珂,陈嘉,余庆,李鹏飞

参考文献(References)：

• [1] XU S F, WANG Y X, WANG Y W, et al. Development and immune evaluation of LAMP1 chimeric DNA vaccine against Singapore grouper iridovirus in orange-spotted grouper,Epinephelus coioides[J]. Fish&Shellfish Immunology,2024, 144:109218.
• [2] LIANG X, LIANG J J, CAO J Q, et al. Oral immunizations with Bacillus subtilis spores displaying VP19 protein provide protection against Singapore grouper iridovirus(SGIV)infection in grouper[J]. Fish&Shellfish Immunology,2023, 138:108860.
• [3] HARIKRISHNAN R, BALASUNDARAM C, HEO M.Fish health aspects in grouper aquaculture[J]. Aquaculture,2011, 320:1-21.
• [4] HUANG L, LI M M, WEI H L, et al. Research on the indirect antiviral function of medicinal plant ingredient quercetin against grouper iridovirus infection[J]. Fish&Shellfish Immunology, 2022, 124:372-379.
• [5] WANG Y X, XU S F, HAN C Z, et al. Curcumin inhibits Singapore grouper iridovirus infection through multiple antiviral mechanisms[J]. Aquaculture, 2022, 562:738870.
• [6] YU Q, LIU M Z, XIAO H H, et al. The inhibitory activities and antiviral mechanism of Viola philippica aqueous extracts against grouper iridovirus infection in vitro and in vivo[J].Journal of Fish Diseases, 2019, 42(6):859-868.
• [7] HUANG C H, ZHANG X B, GIN K Y H, et al. In situ hybridization of a marine fish virus, Singapore grouper iridovirus with a nucleic acid probe of major capsid protein[J]. Journal of Virological Methods, 2004, 117(2):123-128.
• [8] WEI J G, HUANG Y H, ZHU W B, et al. Isolation and identification of Singapore grouper iridovirus Hainan strain(SGIV-HN)in China[J]. Archives of Virology, 2019, 164(7):1869-1872.
• [9] TENG Y, HOU Z W, GONG J, et al. Whole-genome transcriptional profiles of a novel marine fish iridovirus,Singapore grouper iridovirus(SGIV)in virus-infected grouper spleen cell cultures and in orange-spotted grouper,Epinephulus coioides[J]. Virology, 2008, 377(1):39-48.
• [10] LIU Y, TRAN B N, WANG F, et al. Visualization of assembly intermediates and budding vacuoles of Singapore grouper iridovirus in grouper embryonic cells[J]. Scientific Reports, 2016, 6:18696.
• [11] HUANG X H, ZHANG Q Y. Improvement and observation of immunoelectron microscopic method for the localization of frog Rana grylio virus(RGV)in infected fish cells[J].Micron, 2007, 38(6):599-606.
• [12] MILLER D L, PESSIER A P, HICK P, et al. Comparative pathology of ranaviruses and diagnostic techniques[M]//Ranaviruses. Cham:Springer, 2015:171-208.
• [13] YU Q, LIU M Z, WEI S N, et al. Research progress and prospects for the use of aptamers in aquaculture biosecurity[J]. Aquaculture, 2021, 534(1):736257.
• [14] DEHGHANI S, NOSRATI R, YOUSEFI M, et al. Aptamerbased biosensors and nanosensors for the detection of vascular endothelial growth factor(VEGF):a review[J].Biosensors&Bioelectronics, 2018, 110:23-37.
• [15] MOOSAVIAN S A, SAHEBKAR A. Aptamer-functionalized liposomes for targeted cancer therapy[J]. Cancer Letters,2019, 448:144-154.
• [16] ZHANG Y, LAI B S, JUHAS M. Recent advances in aptamer discovery and applications[J]. Molecules, 2019,24(5):941.
• [17] SUN S M, LIU H, HU Y, et al. Selection and identification of a novel ssDNA aptamer targeting human skeletal muscle[J]. Bioactive Materials, 2023, 20:166-178.
• [18] XIAO H H, LIU M Z, LI S Q, et al. Isolation and characterization of a ranavirus associated with disease outbreaks in cultured hybrid grouper(♀Tiger grouper Epinephelus fuscoguttatus×♂Giant grouper E.lanceolatus)in Guangxi,China[J]. Journal of Aquatic Animal Health, 2019, 31(4):364-370.
• [19] YU Q, LIU M Z, WEI S N, et al. Isolation of nervous necrosis virus from hybrid grouper(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂)cultured in Guangxi, China[J].Fish Pathology, 2019, 54(1):16-19.
• [20] YU Q, LIU M Z, XIAO H-H, et al. Selection and characterization of aptamers for specific detection of iridovirus disease in cultured hybrid grouper(Epinephelus Fuscoguttatus♀×E. Lanceolatus♂)[J]. Chinese Journal of Analytical Chemistry, 2020, 48(5):650-661.
• [21] LIU M Z, XIAO H H, ZHANG Q, et al. Antiviral abilities of Curcuma kwangsiensis ingredients against grouper iridoviral infection in vitro and in vivo[J]. Aquaculture Research,2020, 51(1):351-361.
• [22] LI P F, YU Q, ZHOU L L, et al. Probing and characterizing the high specific sequences of ssDNA aptamer against SGIVinfected cells[J]. Virus Research, 2018, 246:46-54.
• [23] YU Q, LIU M Z, XIAO H H, et al. Development of novel aptamer-based enzyme-linked apta-sorbent assay(ELASA)for rapid detection of mariculture pathogen Vibrio alginolyticus[J]. Journal of Fish Diseases, 2019, 42(11):1523-1529.
• [24] MUKHERJEE M, NANDHINI C, BHATT P. Colorimetric and chemiluminescence based enzyme linked apta-sorbent assay(ELASA)for ochratoxin A detection[J]. Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy,2021, 244:118875.
• [25] ZHANG X Y, ZHANG Z M, LI J R, et al. A novel sandwich ELASA based on aptamer for detection of largemouth bass virus(LMBV)[J]. Viruses, 2022, 14(5):945.
• [26] LEE K H, ZENG H Q. Aptamer-based ELISA assay for highly specific and sensitive detection of zika NS1 protein[J]. Analytical Chemistry, 2017, 89(23):12743-12748.
• [27] ZHOU L, LI P, NI S, et al. Rapid and sensitive detection of redspotted grouper nervous necrosis virus(RGNNV)infection by aptamer-coat protein-aptamer sandwich enzymelinked apta-sorbent assay(ELASA)[J]. Journal of Fish Diseases, 2017, 40(12):1831-1838.
• [28] MOJARAD A E, GARGARIA S L M. Aptamer-nanobody based ELASA for detection of Vibrio cholerae O1[J]. Iranian Journal of Microbiology, 2020, 12(4):263-272.
• [29] RASOULINEJAD S, GARGARI S L M. Aptamer-nanobody based ELASA for specific detection of Acinetobacter baumannii isolates[J]. Journal of Biotechnology, 2016, 231:46-54.
• [30] HEGDE A, CHEN C L, QIN Q W, et al. Characterization,pathogenicity and neutralization studies of a nervous necrosis virus isolated from grouper, Epinephelus tauvina,in Singapore.[J]. Aquaculture, 2002, 213:55-72.
• [31] GUO X X, WANG W J, ZHENG Q, et al. Comparative transcriptomic analysis reveals different host cell responses to Singapore grouper iridovirus and red-spotted grouper nervous necrosis virus[J]. Fish&Shellfish Immunology,2022, 128:136-147.
• [32] KIM Y J, CHOI J W. Enzyme-linked aptamer-based sandwich assay(ELASA)for detecting Plasmodium falciparum lactate dehydrogenase, a malarial biomarker[J]. RSC Advances,2022, 12(45):29535-29542.
• [33] WU W H, LI J, PAN D, et al. Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella typhimurium[J]. ACS Applied Materials&Interfaces, 2014, 6(19):16974-16981.
• [34] FAROKHZAD O C, CHENG J J, TEPLY B A, et al. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(16):6315-6320.
• [35] TAN X P, YAN Y, ZHANG G R, et al. Target epidermal damage of Gyrodactylus kobayashii to obtain the effective anthelmintic compound glacial acetic acid[J]. Aquaculture,2023, 577:739993.
• [36] GONG Y M, WEI X F, ZHENG Y Y, et al. Combining phage display technology with In silico-designed epitope vaccine to elicit robust antibody responses against emerging pathogen Tilapia lake virus[J]. Journal of Virology, 2023,97(4):e0005023.