Jiang Xinjie, Huang Yun, Jiang Yi, Yang Guang, Zheng Xiaocong, Gao Shuai
Hainan Laboratory Animal Research Center, Sanya Institute of Hainan Academy of Agricultural Sciences, Sanya 572000, China.
Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Customs, Shenzhen 518045, China.
Viruses. 2025 Jun 20;17(7):872. doi: 10.3390/v17070872.
PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a's collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10 copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management.
猪轮状病毒（PoRV）是仔猪新生腹泻的重要病原体，由于死亡率上升和生产力下降，给全球养猪业造成了巨大经济损失。为满足资源有限地区对便捷快速诊断的迫切需求，我们开发了一种基于CRISPR/Cas12a的检测方法，该方法与重组酶聚合酶扩增（RPA）相结合，用于PoRV的可视化检测。该平台使用优化的RPA引物和crRNA特异性靶向保守的VP6基因，利用Cas12a的附带切割活性通过荧光或侧向流动试纸条（LFD）进行双读出。该检测方法在1小时内检测限为10拷贝/μL，与系统发育相关病原体如传染性胃肠炎病毒（TGEV）无交叉反应。通过消除对热循环仪或专用设备的依赖，该方法可在养猪场、兽医诊所或野外环境中全面应用。侧向流动形式可立即提供比色结果，所需技术专业知识最少，而荧光模式则允许进行半定量分析。本研究为猪群中PoRV的分散监测提供了一个强大且经济高效的平台，满足了资源有限地区对便携式诊断的迫切需求，并加强了兽医健康管理。
