An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and...

Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.