Abstract：
Plant viruses threaten global food security by infecting commercial crops, highlighting the critical need for efficient virus detection to enable timely preventive measures. Current techniques rely on polymerase chain reaction (PCR) for viral genome amplification and require laboratory conditions. This review explores the applications of CRISPR-Cas assisted diagnostic tools, specifically CRISPR-Cas12a and CRISPR-Cas13a/d systems for plant virus detection and analysis. The CRISPR-Cas12a system can detect viral DNA/RNA amplicons and can be coupled with PCR or isothermal amplification, allowing multiplexed detection in plants with mixed infections. Recent studies have eliminated the need for expensive RNA purification, streamlining the process by providing a visible readout through lateral flow strips. The CRISPR-Cas13a/d system can directly detect viral RNA with minimal preamplification, offering a proportional readout to the viral load. These approaches enable rapid viral diagnostics within 30 min of leaf harvest, making them valuable for onsite field applications. Timely identification of diseases associated with pathogens is crucial for effective treatment; yet developing rapid, specific, sensitive, and cost-effective diagnostic technologies remains challenging. The current gold standard, PCR technology, has drawbacks such as lengthy operational cycles, high costs, and demanding requirements. Here we update the technical advancements of CRISPR-Cas in viral detection, providing insights into future developments, versatile applications, and potential clinical translation. There is a need for approaches enabling field plant viral nucleic acid detection with high sensitivity, specificity, affordability, and portability. Despite challenges, CRISPR-Cas-mediated pathogen diagnostic solutions hold robust capabilities, paving the way for ideal diagnostic tools. Alternative applications in virus research are also explored, acknowledging the technology\'s limitations and challenges.
摘要：
植物病毒通过感染商业作物威胁全球粮食安全，强调了高效病毒检测的迫切需要，以便及时采取预防措施。目前的技术依赖于聚合酶链反应(PCR)来进行病毒基因组扩增,并且需要实验室条件。这篇综述探讨了CRISPR-Cas辅助诊断工具的应用，特别是用于植物病毒检测和分析的CRISPR-Cas12a和CRISPR-Cas13a/d系统。CRISPR-Cas12a系统可以检测病毒DNA/RNA扩增子，可以与PCR或等温扩增偶联，允许在混合感染的植物中进行多重检测。最近的研究已经消除了昂贵的RNA纯化的需要，通过横向流动条提供可见的读数来简化过程。CRISPR-Cas13a/d系统可以以最小的预扩增直接检测病毒RNA，提供与病毒载量成比例的读数。这些方法可以在叶片收获后30分钟内进行快速病毒诊断，使它们对现场应用有价值。及时识别与病原体相关的疾病对于有效治疗至关重要;然而，具体,敏感,和具有成本效益的诊断技术仍然具有挑战性。当前的黄金标准，PCR技术,具有操作周期过长等缺点，高成本,和苛刻的要求。在这里，我们更新了CRISPR-Cas在病毒检测中的技术进步，提供对未来发展的见解，多功能应用，和潜在的临床翻译。需要能够实现高灵敏度的田间植物病毒核酸检测的方法。特异性，负担能力，和便携性。尽管面临挑战,CRISPR-Cas介导的病原体诊断解决方案具有强大的功能，为理想的诊断工具铺平道路。还探索了病毒研究中的替代应用，承认技术的局限性和挑战。
