Jaybhaye Siddhant G, Chavhan Rahul L, Hinge Vidya R, Deshmukh Abhijit S, Kadam Ulhas S
Vilasrao Deshmukh College of Agricultural Biotechnology, Nanded Road, Latur, Vasantrao Naik Marathwada Krishi Vidyapeeth, Maharashtra, India.
Plant Molecular Biology and Biotechnology Research Centre (PMBBRC), Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, Gyeongsangnam-do, South Korea.
Virology. 2024 Sep;597:110160. doi: 10.1016/j.virol.2024.110160. Epub 2024 Jun 26.
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.
植物病毒通过感染经济作物威胁全球粮食安全，这凸显了高效病毒检测以采取及时预防措施的迫切需求。当前技术依靠聚合酶链反应（PCR）扩增病毒基因组，且需要实验室条件。本综述探讨了CRISPR-Cas辅助诊断工具的应用，特别是CRISPR-Cas12a和CRISPR-Cas13a/d系统在植物病毒检测与分析中的应用。CRISPR-Cas12a系统可检测病毒DNA/RNA扩增子，可与PCR或等温扩增相结合，实现对混合感染植物的多重检测。最近的研究不再需要昂贵的RNA纯化，通过侧向流动条提供可见读数简化了流程。CRISPR-Cas13a/d系统可在极少预扩增的情况下直接检测病毒RNA，提供与病毒载量成比例的读数。这些方法能在叶片采集后30分钟内实现快速病毒诊断，对现场实地应用很有价值。及时识别与病原体相关的疾病对有效治疗至关重要；然而，开发快速、特异、灵敏且经济高效的诊断技术仍然具有挑战性。当前的金标准PCR技术存在操作周期长、成本高和要求苛刻等缺点。在此，我们更新了CRISPR-Cas在病毒检测方面的技术进展，为未来发展、广泛应用和潜在的临床转化提供见解。需要能够实现高灵敏度、特异性、可承受性和便携性的田间植物病毒核酸检测方法。尽管存在挑战，但CRISPR-Cas介导的病原体诊断解决方案具有强大能力，为理想的诊断工具铺平了道路。还探讨了该技术在病毒研究中的其他应用，同时认识到其局限性和挑战。
