Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA–CRISPR/Cas12a Assay

Abstract

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

“…These challenges drove the development of new real-time PCR and LAMP assays for specific ToBRFV detection [ 31 , 48 ]. While it was shown that CRISPR/Cas12a can be used to detect plant viruses [ 44 ], it was unknown if it could be applied to distinguish between closely related plant viruses of the same genus. Here, we provide evidence that the CRISPR/Cas12a technology can also be applied to identify ToBRFV and distinguish it from the closely related ToMV ( Figure 2 ).…”

“…Recently, CRISPR/Cas12a was applied for the detection of RNA [ 43 , 44 ] and DNA [ 45 ] plant viruses, suggesting its potential application in the detection of plant viral diseases. Here we adopt the CRISPR/Cas12a technology to specifically identify the emerging ToBRFV and to distinguish it from a closely related tobamovirus, ToMV.…”

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

“…Accurate and sensitive virus detection method developments are essential for disease management. The development of CRISPR-Cas-based methods to serve as a diagnostic tool for plant virus detection is emerging, and recently, it has been shown for viruses infecting N. benthamiana and apples ( Aman et al, 2020 ; Jiao et al, 2020 ). Additionally, a CRISPR-Cas method for tomato virus diagnosis was also reported ( Alon et al, 2021 ).…”

“…In plants, CRISPR-Cas system-based virus detection is in the early stages of development. Recently, CRISPR-Cas12a-based assay has been developed for detecting potato virus X (PVX), potato virus Y (PVY), and tobacco mosaic virus (TMV) in the leaf tissues of the model plant Nicotiana benthamiana ( Aman et al, 2020 ), as well as a slightly modified method for detecting viruses in apple leaf samples ( Jiao et al, 2020 ).…”

Application of CRISPR/Cas for Diagnosis and Management of Viral Diseases of Banana

“…Recently, an efficient and rapid RT-RPA-CRISPR/Cas12a system has been developed as a one-step detection assay to diagnose plant RNA viruses ( Aman et al, 2020 ). This diagnostic assay uses a fluorescence visualizer to detect the plant RNA viruses.…”