Onsite detection of plant viruses using isothermal amplification assays

Abstract

Plant diseases caused by viruses limit crop production and quality, resulting in significant losses. However, options for managing viruses are limited; for example, as systemic obligate parasites, they cannot be killed by chemicals. Sensitive, robust, affordable diagnostic assays are needed to detect the presence of viruses in plant materials such as seeds, vegetative parts, insect vectors, or alternative hosts and then prevent or limit their introduction into the field by destroying infected plant materials or controlling insect hosts. Diagnostics based on biological and physical properties are not very sensitive and are time-consuming, but assays based on viral proteins and nucleic acids are more specific, sensitive, and rapid. However, most such assays require laboratories with sophisticated equipment and technical skills. By contrast, isothermal-based assays such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are simple, easy to perform, reliable, specific, and rapid and do not require specialized equipment or skills. Isothermal amplification assays can be performed using lateral flow devices, making them suitable for onsite detection or testing in the field. To overcome non-specific amplification and cross-contamination issues, isothermal amplification assays can be coupled with CRISPR/Cas technology. Indeed, the collateral activity associated with some CRISPR/Cas systems has been successfully harnessed for visual detection of plant viruses. Here, we briefly describe traditional methods for detecting viruses and then examine the various isothermal assays that are being harnessed to detect viruses.

Keywords: CRISPR/Cas-based diagnosis; LAMP; RPA; isothermal amplification; nucleic acid detection; onsite diagnosis.