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.
作者
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
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)
出处
《广东海洋大学学报》
CAS
CSCD
北大核心
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)。
关键词
石斑鱼虹彩病毒
检测
核酸适体
酶联吸附法
grouper iridovirus
detection
aptamer
enzyme-linked apta-sorbent assay(ELASA)
