Department of Biotechnology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India.
Sci Rep. 2021 Jul 9;11(1):14203. doi: 10.1038/s41598-021-93615-9.
Begomoviruses are a major class of Geminiviruses that affects most dicotyledonous plants and causes heavy economic losses to farmers. Early detection of begomovirus is essential to control the spread of the disease and prevent loss. Many available detection methods like ELISA, immunosorbent electron microscopy, PCR or qPCR require expertise in handling sophisticated instruments, complex data interpretation and costlier chemicals, enzymes or antibodies. Hence there is a need for a simpler detection method, here we report the development of a visual detection method based on functionalized gold nanoparticles (AuNP assay). The assay was able to detect up to 500 ag/µl of begomoviral DNA (pTZCCPp3, a clone carrying partial coat protein gene) suspended in MilliQ water. Screening of chilli plants for begomoviral infection by PCR (Deng primers) and AuNP assay showed that AuNP assay (77.7%) was better than PCR (49.4%). The AuNP assay with clccpi1 probe was able to detect begomoviral infection in chilli, tomato, common bean, green gram and black gram plants which proved the utility and versatility of the AuNP assay. The specificity of the assay was demonstrated by testing with total DNA from different plants that are not affected by begomoviruses.
双生病毒是一类重要的 Geminiviruses，会感染大多数双子叶植物，给农民造成严重的经济损失。早期检测双生病毒对于控制疾病的传播和防止损失至关重要。许多现有的检测方法，如 ELISA、免疫吸附电子显微镜、PCR 或 qPCR，都需要专业知识来操作复杂的仪器、复杂的数据解释和更昂贵的化学物质、酶或抗体。因此，需要一种更简单的检测方法，在这里我们报告了一种基于功能化金纳米粒子（AuNP 测定法）的可视化检测方法的开发。该测定法能够检测到悬浮在 MilliQ 水中的多达 500 ag/µl 的双生病毒 DNA（pTZCCPp3，携带部分外壳蛋白基因的克隆）。通过 PCR（Deng 引物）和 AuNP 测定法对辣椒植物进行双生病毒感染的筛选表明，AuNP 测定法（77.7%）优于 PCR（49.4%）。用 clccpi1 探针的 AuNP 测定法能够检测到辣椒、番茄、普通菜豆、绿豆和黑豆植物中的双生病毒感染，证明了 AuNP 测定法的实用性和多功能性。该测定法的特异性通过测试来自不受双生病毒影响的不同植物的总 DNA 得到了证明。
