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利用RPA-CRISPR/Cas13快速、灵敏且可视化检测猪轮状病毒

Rapid, sensitive, and visual detection of porcine rotavirus with RPA-CRISPR/Cas13.

作者信息

Mi Yong, Dai Mingyang, Xing Xiaokun, Zhang Yang, Xu Zhiwen, Zhu Ling

机构信息

College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.

Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, China.

出版信息

Front Microbiol. 2025 Aug 20;16:1617955. doi: 10.3389/fmicb.2025.1617955. eCollection 2025.

Abstract

Porcine rotavirus (PoRV) is one of the major pathogens causing viral enteritis in piglets, posing serious threats to the pig industry and public health. Existing pathogen detection methods, such as RT-qPCR, suffer from complex procedures and strong reliance on equipment, making them difficult to meet the needs of grassroots laboratories or field detection. Therefore, in this study, a novel rapid and visual detection platform, was developed based on the CRISPR/Cas13 system. This platform integrates Recombinase Polymerase Amplification (RPA), T7 transcription, CRISPR trans-cleavage, and fluorescence signal output. Targeting the PoRV VP6 gene, it achieves highly sensitive and specific detection of PoRV without the need for large equipment. The optimized system has a minimum detection limit of 10 copies/μL for standard plasmids, and it was validated with 62 clinical diarrheal samples, yielding results consistent with RT-qPCR, demonstrating good clinical applicability. This platform is simple to operate, intuitive in detection, and cost-effective, making it suitable for rapid field screening of PoRV in resource-limited areas. It provides robust technical support for early diagnosis and epidemiological investigation of porcine rotavirus.

摘要

猪轮状病毒(PoRV)是引起仔猪病毒性肠炎的主要病原体之一,对养猪业和公共卫生构成严重威胁。现有的病原体检测方法,如RT-qPCR,存在操作程序复杂、对设备依赖性强等问题,难以满足基层实验室或现场检测的需求。因此,本研究基于CRISPR/Cas13系统开发了一种新型快速可视化检测平台。该平台整合了重组酶聚合酶扩增(RPA)、T7转录、CRISPR反式切割和荧光信号输出。以PoRV VP6基因为靶点,无需大型设备即可实现对PoRV的高灵敏、特异性检测。优化后的系统对标准质粒的最低检测限为10拷贝/μL,并用62份临床腹泻样本进行验证,结果与RT-qPCR一致,显示出良好的临床适用性。该平台操作简便、检测直观、成本效益高,适用于资源有限地区PoRV的快速现场筛查。它为猪轮状病毒的早期诊断和流行病学调查提供了有力的技术支持。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/87ca/12405341/c9a1232c29e5/fmicb-16-1617955-g001.jpg

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