Location: Sugarbeet and Potato ResearchTitle: CRISPR-based isothermal next-generation diagnostic method for virus detection in sugarbeet
Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2021
Publication Date: 7/8/2021
Citation: Ramachandran, V., Weiland, J.J., Bolton, M.D. 2021. CRISPR-based isothermal next-generation diagnostic method for virus detection in sugarbeet. Frontiers in Microbiology. 12. Article e679994. https://doi.org/10.3389/fmicb.2021.679994.
Interpretive Summary: Sugarbeet is a major sucrose-contributing source for sugar industries in the United States. Many different disease-causing agents infect sugarbeet, including the Beet necrotic yellow vein virus (BNYVV) which causes the devastating ‘Rhizomania’ disease. Diagnostic methods to accurately identify BNYVV are essential for evaluating field soil and plants to manage the disease. Recent advancements in molecular biology led to the development of a method called clustered regularly interspaced short palindromic repeats (CRISPR) for virus detection. Methods that rely on CRISPR technology have been shown to be highly sensitive and accurate for human virus diagnostics, including Coronavirus and Zika Virus. In this study, we report the development of such a CRISPR-based method for BNYVV detection in sugarbeet roots. This newly developed detection method is advantageous in terms of potentially providing BNYVV detection under field conditions and sets the stage for the development of viral diagnostics in sugarbeet and other important world crops.
Technical Abstract: Rhizomania is a disease of sugarbeet caused by Beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil is necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of CRISPR-Cas12a based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Towards this end, we have developed a reverse-transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively the reporter signal. To our knowledge, this is the first report establishing an isothermal RT-RPA and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.