基于核酸适体LYGV1c的酶联吸附法检测石斑鱼虹彩病毒感染  

Detection of Grouper Iridovirus by Enzyme-linked Adsorption Method Based on Aptamer LYGV1c

作　　者：刘明珠 黄静 程远 韦云依 牟容丽 黄琳 竺利波[1] 陆兰天[1] 柯珂[1] 陈嘉 余庆 李鹏飞 LIU Mingzhu;HUANG Jing;CHENG Yuan;WEI Yunyi;MU Rongli;HUANG Lin;ZHU Libo;LU Lantian;KE Ke;CHEN Jia;YU Qing;LI Pengfei(Guangxi Key Laboratory of Aquatic Biotechnology and Modern Ecological Aquaculture,Guangxi Engineering Research Center for Fishery Major Diseases Control and Efficient Healthy Breeding Industrial Technology,Guangxi Academy of Sciences,Nanning 530007,China;College of Food and Quality Engineering,Nanning College,Nanning 530200,China;China-ASEAN Modern Fishery Industry Technology Transfer Demonstration Center,Beibu Gulf Marine Industry Research Institute,Guangxi Academy of Sciences,Nanning 530007,China)

机构地区：[1]广西水产生物技术与现代生态养殖重点实验室,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,广西科学院,广西南宁530007 [2]南宁学院食品与质量工程学院,广西南宁530200 [3]中国-东盟现代渔业产业技术转移示范中心,广西科学院北部湾海洋产业研究院,广西南宁530007

出　　处：《广东海洋大学学报》2024年第3期1-8,共8页Journal of Guangdong Ocean University

基　　金：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)。

摘　　要：【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。【Objective】To develop the aptamer LYGV1c-based enzyme-linked apta-sorbent assay(LYGV1c-ELASA)for accurate and rapid detection of Singapore grouper iridovirus Guangxi strain(SGIV-Gx),and to provide theoretical support for the development of rapid detection system technology in aquaculture.【Method】An aptamer enzyme-linked adsorption method was developed based on biotin-labeled LYGV1c.The specificity,sensitivity and stability of bio-LYGV1c were analyzed in SGIV-Gx infection.Hybrid Grouper(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂)were infected by SGIV-Gx,and the relative expression of MCP of SGIV-Gx was determined by fluorescence quantitative PCP(qPCR),the results of qPCR were compared with that of LYGV1c-ELASA to verify the reliability of ELASA.【Result】LYGV1c-ELASA could detect SGIV infection with high specificity,and the working concentration was 500 nmol/L,incubation was 20 min,the suitable binding temperature was 4‒28℃,and the detection limit was 5×103 cells/mL.The results of infection test showed that the OD450 value of SGIV-Gx increased with the increase of virus concentration in vivo.The qPCR result was the same as LYGV1c-ELASA detection results in vivo.【Conclusion】LYGV1c-ELASA technology can be used for the detection of SGIV-Gx in vitro and in vivo.LYGV1c-ELASA is a technique for rapid diagnosis and real-time monitor of SGIV-Gx infection.

关 键 词：石斑鱼虹彩病毒 检测 核酸适体 酶联吸附法 

分 类 号：S941.41[农业科学—水产养殖]