A frontline, rapid, nucleic acid-based Fusarium circinatum detection system using CRISPR/Cas12a combined with recombinase polymerase amplification

Pitch canker caused by the fungus Fusarium circinatum is a damaging disease that affects pines in Europe, South Africa, and North America in both the southeast and west coast of the USA. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Timely detection, an important pillar of the quarantine effort, can efficiently prevent the introduction of F. circinatum into new areas or facilitate management and eradication strategies in already infested sites. In this study, we developed a F. circinatum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPA-CRISPR/Cas12a). After obtaining DNA, this novel method can be utilized for the molecular identification of F. circinatum using the naked eye, and can specifically detect F. circinatum at DNA concentrations as low as 200 fg within 20 min at 37 °C. The system is sensitive for both standard laboratory samples and samples from the field. In summary, we have developed a simple, rapid, sensitive, unaided-eye visualization, RPA-CRISPR/Cas12a-based detection system for the molecular identification of F. circinatum that does not require technical expertise or expensive ancillary equipment.