Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection

ABSTRACT Implementing effective monitoring strategies is fundamental to protect crops from pathogens and to ensure the food supply as the world population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become pandemic in less than a decade. Here we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, while no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a reference real-time quantitative PCR test widely used, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field-applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it still may be useful to confirm ToBRFV, detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge this is the first field-compatible CRISPR-based test to detect ToBRFV, which combines isothermal amplification with a simplified nucleic acid extraction protocol.