An RPA‐CRISPR/Cas12a based platform for rapid, sensitive, and visual detection of Apis mellifera filamentous virus

Apis mellifera filamentous virus (AmFV) is an emerging DNA virus significantly affecting honey bee health. AmFV infections weaken bee resistance to other pathogens, and can cause tissue lysis and death. Early, accurate detection of AmFV is crucial for timely intervention and preventing large-scale outbreaks. Current AmFV detection relies largely on polymerase chain reaction (PCR)-based methods. To enable rapid field detection of AmFV, we developed a rapid and ultrasensitive detection platform using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 12a (Cas12a) technology. A CRISPR RNA (crRNA1) specifically targeting the AmFV Bro gene was designed, ensuring no cross-reactivity with other insect DNA viruses or uninfected honey bees. After optimization of the reaction time, the platform generated results within 35 min: 20 min for the RPA reaction and 15 min for CRISPR-mediated cleavage. Two visualization approaches, fluorescence-based and lateral flow dipstick, were used to display the detection results. The detection sensitivity of both approaches was as few as 10 copies of the AmFV genome. Validation with field-collected honey bee samples demonstrated consistency with conventional PCR, revealing widespread latent AmFV infections in the field. Taken together, we successfully developed an RPA-CRISPR/Cas12 platform for rapid, specific, and sensitive detection of AmFV in Apis mellifera and Apis cerana. This platform holds promise as a simple, accurate, and cost-effective tool for point-of-care AmFV diagnosis in the field.