A rapid and sensitive RPA-Cas12a assay for detection of banana bunchy top virus (Babuvirus musae)

Abstract

Banana bunchy top virus (BBTV, Babuvirus musae ) is one of the most damaging viral threats to banana production, yet frontline diagnostics rely on PCR, ELISA or proprietary assays that are poorly suited to low resource deployment or associated with high per test cost. We developed a recombinase polymerase amplification (RPA)-Cas12a assay that achieves high sensitivity using crude extracts with fluorescence or lateral flow readouts. Using RPA primers to BBTV DNA-R gene and a Cas12a crRNA targeting a conserved site, we developed the first CRISPR-based diagnostic assay for BBTV. The assay was configured in a one-tube workflow in which the RPA and Cas12a components are separated until amplification is complete, reducing contamination risk. With a DNA-R gBlock standard, the one-tube assay achieved a limit of detection of 5.11 aM (~ 3 copies/µl) in both formats, which is around 250-fold more sensitive than a commercially available fluorescence RPA and about fourfold more sensitive than an available RPA-based lateral flow. The RPA-Cas12a assay reliably detected BBTV in both fresh and dried leaf material, across isolates from both genetic subgroups, and showed no cross-reactivity with common banana viruses. A two-step high sensitivity workflow further reduced the limit of detection to near single copy levels. This work establishes a generic RPA-Cas12a framework for plant virus diagnostics and an immediately deployable assay for BBTV surveillance, eradication and clean planting material programmes.