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重组酶聚合酶扩增技术结合侧流层析试纸条快速检测新加坡石斑鱼虹彩病毒

潘莹 ,  杨家辉 ,  彭发永 ,  黄友华 ,  秦启伟 ,  黄晓红

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文章导航 > 水产学报 > 2024 > 48(5): 059411
潘莹, 杨家辉, 彭发永, 黄友华, 秦启伟, 黄晓红. 2024. 重组酶聚合酶扩增技术结合侧流层析试纸条快速检测新加坡石斑鱼虹彩病毒. 水产学报, 48(5): 059411. doi: 10.11964/jfc.20231014187
引用本文: 潘莹, 杨家辉, 彭发永, 黄友华, 秦启伟, 黄晓红. 2024. 重组酶聚合酶扩增技术结合侧流层析试纸条快速检测新加坡石斑鱼虹彩病毒. 水产学报, 48(5): 059411. doi: 10.11964/jfc.20231014187
shu
PAN Ying, YANG Jiahui, PENG Fayong, HUANG Youhua, QIN Qiwei, HUANG Xiaohong. 2024. Rapid detection of Singapore grouper iridovirus by a recombinase polymerase amplification combined with lateral flow dipstick. Journal of Fisheries of China, 48(5): 059411. doi: 10.11964/jfc.20231014187
Citation: PAN Ying, YANG Jiahui, PENG Fayong, HUANG Youhua, QIN Qiwei, HUANG Xiaohong. 2024. Rapid detection of Singapore grouper iridovirus by a recombinase polymerase amplification combined with lateral flow dipstick. Journal of Fisheries of China, 48(5): 059411. doi: 10.11964/jfc.20231014187
shu

重组酶聚合酶扩增技术结合侧流层析试纸条快速检测新加坡石斑鱼虹彩病毒

    • 1.

      华南农业大学海洋学院，广东 广州　510642

    • 2.

      广州华农南沙渔业研究院，广东 广州　511464

  • 基金项目:
    国家重点研发计划课题 (2023YFD2401703)；国家海水鱼产业技术体系专项 (CARS-47-G16)；国家自然科学基金 (U20A20102)
详细信息
    作者简介:

    潘莹 (照片)，从事水生动物病害控制研究，E-mail：panying@stu.scau.edu.cn

    通讯作者: 黄晓红，从事水生动物疾病防治研究，E-mail：huangxh@scau.edu.cn

  • 中图分类号: S 942

Rapid detection of Singapore grouper iridovirus by a recombinase polymerase amplification combined with lateral flow dipstick

    • 1.

      College of Marine Sciences, South China Agricultural University, Guangzhou　510642, China

    • 2.

      Nansha-South China Agricultural University Fishery Research Institue Guangdong, Guangzhou　511464, China

  • Fund Project: National Key Research and Development Program of China (2023YFD2401703); China Agriculture Research System of MOF and MARA (CARS-47-G16); National Natural Science Foundation of China (U20A20102)
More Information

    摘要
  • 为建立一种快速灵敏、可视化的适用于临床样品检测新加坡石斑鱼虹彩病毒 (Singapore grouper iridovirus，SGIV)的方法，本研究针对SGIV特异基因ORF014L序列设计特异性引物及探针，建立重组酶聚合酶扩增 (recombinase polymerase amplification，RPA)技术及结合侧流层析试纸条 (lateral flow dipstick，LFD) (RPA-LFD)的SGIV检测技术。RPA反应使用10 μmol/L的引物浓度，在40.1 °C恒温反应20 min即可完成特异性病毒的检测，最低检测限为102 个/μL标准质粒。RPA-LFD反应在42 °C恒温反应8 min可将检测结果通过试纸条可视化呈现，最低检测限为101 个/μL标准质粒，且不与其他常见水生动物病原发生交叉反应，临床样品检测结果也与PCR检测结果一致。RPA、RPA-LFD均能特异性检测SGIV，两者的检测限均比常规PCR灵敏。RPA-LFD法具有快捷简单、结果可视化的特点，在临床应用具有较好的应用前景。
    Singapore grouper iridovirus (SGIV), a novel species of Ranavirus, caused more than 90% mortality in larval and juvenile groupers. Up to now, there is still a lack of effective prevention and control strategies for SGIV. Therefore, it is essential to develop convenient diagnostic methods for filed detection of SGIV without special equipment. In this study, to establish a rapid, sensitive and visualized method for the detection of SGIV in clinical samples, specific primers and probes were designed by targeting the SGIV ORF014L, and a recombinase polymerase amplification (RPA) technique combined with lateral flow dipstick (LFD) (RPA-LFD) was developed for the detection of SGIV. The results showed that the RPA reaction specifically detected target fragment of SGIV within 20 min at 40.1 °C with the lowest detection limit of 102 copies/μL. The RPA-LFD reaction at a constant temperature of 42 °C for 8 min was able to visualize the results on the test strips with the lowest detection limit of 101 copies/μL, and showed no cross-reaction with other common aquatic pathogens. The coincidence rate of positive test of clinical samples was consistent between RPA-LFD and PCR methods. Both RPA and RPA-LFD could specifically detect SGIV with lower limit than conventional PCR assay. Taken together, RPA-LFD assay developed in the present study provides a convenient, specific, sensitive and visualized method for on-site rapid detection of SGIV without special equipment.
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  • 图 1  不同RPA引物对的扩增结果

    Figure 1.  Amplification results of different RPA primer pairs

    下载: 全尺寸图片 幻灯片

    图 2  RPA检测方法的反应温度(a)、反应时间(b)、引物浓度(c)优化结果

    Figure 2.  Optimization results of different temperatures (a), time (b) and concentrations of primer pair (c) for RPA assay

    下载: 全尺寸图片 幻灯片

    图 3  不同RPA-LFD引物探针组合的扩增结果

    Figure 3.  Amplification results of different RPA-LFD primers and probes

    下载: 全尺寸图片 幻灯片

    图 4  RPA-LFD检测方法的反应温度(a)、反应时间(b)优化结果

    Figure 4.  Optimization results of different temperatures(a) and time(b) for RPA-LFD assay

    下载: 全尺寸图片 幻灯片

    图 5  RPA(a)、 RPA-LFD(b)检测方法的特异性结果

    Figure 5.  Specificity results of RPA (a) and RPA-LFD (b) assays

    下载: 全尺寸图片 幻灯片

    图 6  PCR(a)、RPA(b)、 RPA-LFD(c)检测方法的灵敏性结果

    Figure 6.  Sensitivity results of PCR (a), RPA (b) and RPA-LFD (c) assays

    下载: 全尺寸图片 幻灯片

    图 7  PCR法(a)(b)、RPA-LFD(c)(d)法的临床样品检测结果

    Figure 7.  Results of clinical samples tested by PCR (a)(b) and RPA-LFD (c)(d) assays

    下载: 全尺寸图片 幻灯片

    表 1  实验相关引物及探针序列信息

    Table 1.  Sequence information of primers and probes used in this study

    名称
    name    		序列 (5′-3′)
    sequence(5′-3′)
    014-104FTCCGACTATCAATCAAACGTCATCGCCTCG
    014-104R

    014-165F    		CACCCGTTGTCGCAGTTTCGTATAGACCC

    CAAGTGACGACCGAACACCGGCTACCAGC
    014-165RAGCCATCGAACCCGTAGTCATATTGTGGA
    014-271FGCTACCAGCATCCAATTCTCACGCAAGAT
    014-271RGGCGATGACGTTTGATTGATAGTCGGAAA
    014-348F

    014-348R    		GCTACCAGCATCCAATTCTCACGCAAGAT

    CACCCGTTGTCGCAGTTTCGTATAGACCC
    probe(FAM)-AGCATCCAATTCTCACGCAAGATGGCCCGT-
    (THF)-GAAGTCAGTTTCTATTC-(C3spacer)
    R1(biotin)-CACCCGTTGTCGCAGTTTCGTATAGACCC
    R2(biotin)-AGGCGATGACGTGTGATTGATAGTCTGAAA
    R3(biotin)-CGCATGTCGACCGAGGCGATGACGTTTGAT
    PCR-266FAGCACAAGTTTCCTCCCG
    PCR-266RACACCCGTTGTCGCAGTT
    pcDNA3.1-3×HA-ORF014-FCTTGGTACCGAGCTCATGTATAGAGGATTTTCTTTAA
    pcDNA3.1-3×HA-ORF014-RGCCCTCTAGACTCGA TATGTACACCCGTTGTC
    下载: 导出CSV
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