首页|基于核酸适体LYGV1c的酶联吸附法检测石斑鱼虹彩病毒感染

基于核酸适体LYGV1c的酶联吸附法检测石斑鱼虹彩病毒感染

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  • 国家科技期刊平台
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  • NSTL
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[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。
Detection of Grouper Iridovirus by Enzyme-linked Adsorption Method Based on Aptamer LYGV1c
[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.

grouper iridovirusdetectionaptamerenzyme-linked apta-sorbent assay(ELASA)

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

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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

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

南宁学院食品与质量工程学院,广西 南宁 530200

石斑鱼虹彩病毒 检测 核酸适体 酶联吸附法

国家自然科学基金广西自然科学基金广西自然科学基金广西自然科学基金国家重点研发计划现代农业产业技术体系建设项目广西创新团队项目

323731752023JJG1300072022GXNSFBA0355212022JJA1300742022YFD2401200nycytxgxcxtd-2021-08-02

2024

10.3969/j.issn.1673-9159.2024.03.001

广东海洋大学学报
广东海洋大学

广东海洋大学学报

CSTPCDCHSSCD北大核心
影响因子：0.444
ISSN：1673-9159
年,卷(期)：2024.44(3)
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