Development of a Recombinase Polymerase Amplification–CRISPR/Cas12a Detection System for Cacao Mild Mosaic Virus

Plant viruses that cause minimal to no disease symptoms may not support readily detectable virus levels. Such viruses are of concern when they persist in plant germplasm collections or in breeding populations because they can provide an inoculum that can be spread and potentially cause outbreaks in susceptible plant species. The mealybug-transmitted cacao mild mosaic virus (CaMMV) causes symptomatic and asymptomatic infection of cacao trees that varies seasonally. The virus accumulates to low levels in leaves and petioles of at least some cacao genetic groups, which has confounded reliable CaMMV detection. Here, a multiplex recombinase polymerase amplification (RPA) assay was developed to increase the reliability of CaMMV detection. Three RPA primers were designed to amplify two regions of the movement protein ( mp) gene of CaMMV, yielding fragments of 362 and 284 bp. To increase the detection sensitivity and specificity of CaMMV, two guide RNAs (20 bp) targeting both the CaMMV RPA amplicons were designed to activate Cas12a-mediated collateral cleavage of a fluorescent reporter. An RPA detection efficiency of 100% was achieved with respect to six known CaMMV mp variants, and the analytical sensitivity ranged from approximately 3 to 40 detectable CaMMV genomes. No signal was observed when cloned cacao-infecting badnavirus sequences or virus-free cacao were used as the template, indicating that this assay is highly specific for CaMMV.