基于RAA-CRISPR/Cas13a的鲤春病毒血症病毒快速检测方法的建立
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作者单位:

1.天津农学院水产学院 天津 300384 ;2.深圳海关动植物检验检疫技术中心 广东 深圳 518045 ;3.中国水产科学研究院南海水产研究所 广东 广州 510300

作者简介:

张磊,E-mail:2667267852@qq.com

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中图分类号:

S943

基金项目:

海关总署科研项目(2024HK026; 2022HK007)和广东省“生物安全技术”专项重点研发项目(2022B1111030001)共同资助


Rapid Detection Method for Spring Viremia of Carp Virus Based on RAA-CRISPR/Cas13a
Author:
Affiliation:

1.College of Fisheries, Tianjin Agricultural University, Tianjin 300384 , China ;2.Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Centre, Shenzhen 518045 , China ;3.South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300 , China

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    摘要:

    鲤春病毒血症病毒(spring viremia of carp virus, SVCV)是一种高致病性病原,对鲤科鱼类造成巨大危害,被世界动物卫生组织列为必须通报疫病。2002 年,鲤春病毒血症(spring viremia of carp, SVC)在中国首次检出后,迅速在全国蔓延,给我国鲤鱼养殖业造成巨大威胁。因此,我国农业农村部将其列为二类动物疫病。迄今为止,早期、快速、准确的诊断仍然是控制其传播和流行的重要手段。本研究基于 CRISPR/Cas13a 系统结合重组酶介导等温核酸扩增技术(recombinase-aided amplification, RAA),通过对 GenBank 上登录的 SVCV 全基因序列比对,针对 SVCV 聚合酶 L 基因高度保守的区域设计了一组 RAA 扩增引物和相应 crRNA;建立了一种可用于 SVCV 现场快速检测的 RAA-CRISPR/Cas13a 的方法,可以覆盖 SVCV 的 4 种不同基因型(Ia、Ib、IcId)。按照世界动物卫生组织推荐的检测方法验证程序,经实验验证该检测方法有较好的重复性,最低检出限为 115 copies/μL,与其他病原之间不发生交叉反应。通过对实验室分离保存的 60 份来自进出境水生动物疫病监测、国内重大水生动物疫病监测以及攻毒实验的样本进行检测,RAA-CRISPR/Cas13a 检测结果与实时荧光定量 PCR、套式 RT-PCR 和病毒分离培养结果完全一致,诊断灵敏度和诊断特异性均达到 100%。采用本研究建立的方法在 4 个不同实验室开展比对,在相同条件下对 20 份样本进行检测,4 个实验室的检测结果一致。结果表明,本研究建立的方法重现性良好,为首次采用 CRISPR-Cas13a 系统进行 SVCV 检测的研究,对 SVCV 快速诊断和防控具有重要现实意义。

    Abstract:

    Spring viremia of carp virus (SVCV) is highly pathogenic in cyprinid fishes, and spring viremia of carp (SVC) has been listed as a notifiable disease by the World Organization for Animal Health. Spring viremia of common carp (Cyprinus carpio) is a viral infectious disease prevalent in Europe, Asia, and North America. In 2002, SVC was detected for the first time in China and spread rapidly throughout the country, posing a huge threat to the carp farming industry in China. It was listed as a second-class animal disease. So far, early, rapid, and accurate diagnosis remains an important means to control its spread and prevalence. At present, commonly used methods to detect SVCV require specific amplification equipment and temperature cycles and are prone to false negatives. Moreover, they have high equipment and personnel requirements and a long detection cycle. Fast and effective daily testing is also difficult to complete. Recombinase-aided amplification (RAA) is a rapid amplification technique of nucleic acid at constant temperature, which can achieve nucleic acid amplification at constant temperature and is easy to operate. Regular clustered interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) systems, such as CRISPR-Cas12 and CRISPR-Cas13, are combined with isothermal amplification to improve the specificity and sensitivity of RNA virus detection. This technology is characterized by constant temperature, fast reaction speed, and miniaturization, and it is suitable for rapid diagnosis and regular monitoring in the field. As an SVC international reference laboratory recognized by WOAH, this research team has been cooperating with the SVC reference laboratory in the UK to carry out the screening, comparison, and optimization of nucleic acid rapid detection. Based on the CRISPR/Cas13a system and recombinase-mediated isothermal nucleic acid amplification technology, RAA amplification primers and corresponding crRNA primers were designed for the highly conserved region of SVCV polymerase L gene by aligning the whole gene sequence of SVCV registered on GenBank, and a preliminary RAA-CRISPR/Cas13a detection method was established for the rapid detection of SVCV in the field. It can cover four genotypes of SVCV (Ia, Ib, Ic, Id). In accordance with the detection and verification procedures recommended by the World Organization for Animal Health, results confirmed that some of the detection results were reproducible, the minimum detection concentration was 115 copies/μL, and it did not cross-react with other pathogens. The detection of 60 samples isolated and stored in the laboratory for the monitoring of inbound and outbound aquatic animal diseases, the monitoring of major aquatic animal diseases in China, and the challenge experiment indicated that the results of RAA-CRISPR/Cas13a detection are consistent with those of nested fluorescent RT-PCR. The results of RT-PCR and virus isolation and culture were also consistent, and the diagnostic sensitivity and specificity were both 100%. Under the same conditions, 20 samples were tested in four laboratories, and the results of the four laboratories were consistent. The results proved the good reproducibility of the study. This study is the first to use the CRISPR-Cas13a system for SVCV detection. The developed method could be applied in the rapid diagnosis and prevention of SVCV.

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张磊, 王津津, 廖立珊, 薛霖朗, 吴江, 朱鹏, 张子怡, 辛卓润, 朱裕敏, 孙敬锋, 姜敬哲, 刘荭, 孙洁. 基于 RAA-CRISPR/ Cas13a 的鲤春病毒血症病毒快速检测方法的建立. 渔业科学进展, 2025, 46(6): 241–248

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  • 收稿日期:2025-01-13
  • 最后修改日期:2025-02-10
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  • 在线发布日期: 2025-11-10
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