Niño-Jimenez Diana-Patricia, López-López Karina, Cuervo-Ibáñez Maritza
International Center for Tropical Agriculture (CIAT), Germplasm Health Unit (GHU), Palmira, Valle del Cauca, Colombia.
Universidad Nacional de Colombia, Palmira, Valle del Cauca, Colombia.
Heliyon. 2024 Mar 5;10(6):e27604. doi: 10.1016/j.heliyon.2024.e27604. eCollection 2024 Mar 30.
Cassava ( Crantz) is a crop of global economic and food safety importance, used for human consumption and in various industrial applications. The genebank of the Genetic Resources Program of the Alliance of Bioversity International and CIAT currently holds the world's largest cassava collection, with 5965 accessions from 28 countries. Managing this extensive collection involves indexing quarantine pathogens as a phytosanitary certification requirement for safely distributing cassava germplasm. The study therefore aimed to optimize a quantitative diagnostic protocol to detect cassava common mosaic virus (CsCMV) using quantitative PCR (qPCR) as a better alternative to other molecular techniques. This was done through designing primers and a probe in the RdRP region of CsCMV, and optimizing the qPCR conditions of the diagnostic protocol using primer concentration assays, and reaction amplification conditions such as volume and reaction time. We also evaluated the qPCR protocol by comparing the results of 140 cassava accession evaluations using three diagnostic methodologies (DAS-ELISA, end-point PCR, and qPCR) for CsCMV. Our protocol established that qPCR technique analysis is ten-times more sensitive in detecting CsCMV compared to end-point PCR, showing a maximum detection level of 77.97 copies/μL of plasmid, with 76 min of reaction time. The comparison allowed us to verify the level of CsCMV detection through the techniques evaluated, concluding that qPCR was more sensitive and allowed the quantification of viral concentration. The optimized qPCR protocol will be used to accelerate diagnostic screening of cassava germplasm for the presence or absence of CsCMV to ensure safe movement and distribution of disease-free germplasm.
木薯（Crantz）是一种具有全球经济和食品安全重要性的作物，可用于人类消费及各种工业应用。国际生物多样性联盟和国际热带农业中心遗传资源项目的基因库目前保存着世界上最大的木薯种质收集品，包含来自28个国家的5965份种质。管理这一庞大的收集品涉及对检疫性病原体进行索引，这是安全分发木薯种质的植物检疫认证要求。因此，该研究旨在优化一种定量诊断方案，使用定量聚合酶链反应（qPCR）检测木薯普通花叶病毒（CsCMV），作为比其他分子技术更好的替代方法。这是通过在CsCMV的RNA依赖的RNA聚合酶（RdRP）区域设计引物和探针，并使用引物浓度测定以及反应体积和反应时间等反应扩增条件来优化诊断方案的qPCR条件实现的。我们还通过比较使用三种诊断方法（双抗夹心酶联免疫吸附测定法、终点聚合酶链反应和qPCR）对140份木薯种质进行CsCMV评估的结果，对qPCR方案进行了评估。我们的方案表明，与终点聚合酶链反应相比，qPCR技术分析检测CsCMV的灵敏度高10倍，在反应时间为76分钟时，质粒的最大检测水平为77.97拷贝/微升。通过该比较，我们能够验证通过所评估技术检测CsCMV的水平，得出qPCR更灵敏且能够对病毒浓度进行定量的结论。优化后的qPCR方案将用于加速对木薯种质是否存在CsCMV的诊断筛选，以确保无病种质的安全转移和分发。
