CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

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 are 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 a 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. Toward 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 target sequence. 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 through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13

“…Our results indicate that nucleic acid amplification by RT-PCR or RT-RPA coupled with CRISPR-Cas12a targeting is a suitable strategy to detect RNA viruses from infected plant tissues in a simple and effective manner, extending previous work. [15][16][17]28 In particular, we show that accurate detection can be performed in 40 min starting from harvested leaves at a low constant temperature of 37−40 °C, with no RNA purification by using an alkaline lysis solution and with a visual readout using lateral flow strips. The use of crude extracts for nucleic acid detection represents an ideal approach that is expected to gain momentum, provided that it is tested in widely different plants.…”

Onsite detection of plant viruses using isothermal amplification assays

“…RT‐RPA followed by CRISPR/Cas12a‐based detection using real‐time fluorescence was also used to detect beet necrotic yellow vein virus (BNYVV) (Ramachandran et al ., 2021). Jiao et al .…”

“…RT-RPA followed by CRISPR/Cas12a-based detection using realtime fluorescence was also used to detect beet necrotic yellow vein virus (BNYVV) (Ramachandran et al, 2021). Jiao et al (2021) performed CRISPR/Cas12a-based detection of viruses infecting apples, such as apple necrotic mosaic virus, apple stem pitting virus, apple stem grooving virus, apple chlorotic leaf spot virus, and apple scar skin viroid (Table 1).…”

Antiviral strategies: What can we learn from natural reservoirs?

“…For example, the paper‐based lateral‐flow assay and magnetoelastic sensing system can be easily used to detect viruses in the field without any expensive equipment; results are obtained in minutes, avoiding the time and difficulty associated with transferring samples to a laboratory for ELISA or PCR (Guo et al, 2016; Nguyen et al, 2020). Recombinase polymerase amplification and CRISPR‐based virus detection systems have also been established to rapidly, sensitively, and specifically detect viruses in humans and plants (Babu et al, 2018; Ramachandran et al, 2021). We believe that the application of these new techniques will promote fast and accurate viral reservoir detection in the plant kingdom.…”