基于CRISPR/Cas12a的牛病毒性腹泻病毒现地快速诊断平台的建立

doi:10.1016/j.jia.2024.03.074

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (8): 2872-2876.DOI: 10.1016/j.jia.2024.03.074

基于CRISPR/Cas12a的牛病毒性腹泻病毒现地快速诊断平台的建立

收稿日期:2023-10-18 接受日期:2024-01-20 出版日期:2024-08-20 发布日期:2024-07-29

A CRISPR/Cas12a-based platform for rapid on-site bovine viral diarrhea virus diagnostics

Meixi Wang^1,3*, Jitao Chang^1,4*, Yuxin Han¹, Chaonan Wang¹, Songkang Qin¹, Jun Wang¹, Lulu Zhang², Yuanmao Zhu¹, Fei Xue¹, Fang Wang¹, Hongliang Chai³, Yulong Wang^3#, Xinjie Wang^2#, Xin Yin^1#

¹State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China
²Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
³ College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China
⁴ Institute of Western Agriculture, Chinese Academy of Agricultural Sciences, Changji 831100, China

Received:2023-10-18 Accepted:2024-01-20 Online:2024-08-20 Published:2024-07-29
About author:Meixi Wang, E-mail: m13582720162@163.com; Jitao Chang, E-mail: changjitao@caas.cn; #Correspondence Yulong Wang, E-mail: 113659770@qq.com; Xinjie Wang, E-mail: wangxinjie@caas.cn; Xin Yin, E-mail: yinxin@caas.cn * These authors contributed equally to this study.
Supported by:
This study was supported by the Inner Mongolia Autonomous Region Major Science and Technology Project, China(2020ZD0006 and 2021ZD0013), the Natural Science Foundation of Heilongjiang Province, China (JQ2021C005), the National Key Research and Development Plan of China (2022YFE0140700), and the National Natural Science Foundation of China (32172940).

摘要/Abstract

摘要：

牛病毒性腹泻（BVD）是由牛病毒性腹泻病毒（BVDV）引起的一种在世界范围内广泛流行的传染病。牛感染BVDV后导致产奶量下降、产肉质量降低及繁殖能力下降，给全球畜牧业带来巨大负担。BVDV感染妊娠期母畜后，可通过胎盘传播至胎儿。若胎儿幸存则成为持续感染（PI）牛，其分泌物和排泄物中存在大量病毒，是BVDV的主要传染源之一。因此，快速准确的诊断对于BVD预防控制，尤其是持续感染牛的净化剔除至关重要。基于此，本研究开发了一种基于CRISPR/Cas12a的BVDV检测方法，用于BVDV的快速现场诊断。首先，通过序列比对和筛选，获得6个BVDV特异crRNA，且通过组合筛选获得可检测当前所有BVDV流行毒株的crRNA混合物；同时，优化设计了RT-ERA特异性扩增反应体系，而后将其与CRISPR-Cas12a系统相结合，建立了一种BVDV结果可视的检测方法。该方法具有高灵敏度和特异性，能在45分钟内检测低至20个拷贝的病毒量，且不与其它共感染的病原体发生交叉反应。此外，该检测方法可识别在中国流行的所有已知的BVDV-1和BVDV-2亚型毒株。在临床样品检测中发现，该检测方法与RNA试剂盒提取样本的荧光定量PCR（qPCR）结果一致，对释放剂释放RNA样本的检测敏感性更高。总之，本研究建立的基于Cas12a的快速、灵敏度高、特异强，不依赖实验室精密仪器，且可得到可视化检测结果的BVDV现地检测方法，该方法的应用对于BVDV精准防控具有重要实践意义。

Meixi Wang, Jitao Chang, Yuxin Han, Chaonan Wang, Songkang Qin, Jun Wang, Lulu Zhang, Yuanmao Zhu, Fei Xue, Fang Wang, Hongliang Chai, Yulong Wang, Xinjie Wang, Xin Yin. 基于CRISPR/Cas12a的牛病毒性腹泻病毒现地快速诊断平台的建立[J]. Journal of Integrative Agriculture, 2024, 23(8): 2872-2876.

Meixi Wang, Jitao Chang, Yuxin Han, Chaonan Wang, Songkang Qin, Jun Wang, Lulu Zhang, Yuanmao Zhu, Fei Xue, Fang Wang, Hongliang Chai, Yulong Wang, Xinjie Wang, Xin Yin. A CRISPR/Cas12a-based platform for rapid on-site bovine viral diarrhea virus diagnostics[J]. Journal of Integrative Agriculture, 2024, 23(8): 2872-2876.

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基于CRISPR/Cas12a的牛病毒性腹泻病毒现地快速诊断平台的建立

A CRISPR/Cas12a-based platform for rapid on-site bovine viral diarrhea virus diagnostics

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