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Updated: May 21, 2025
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
An RPA-CRISPR/Cas12a based platform for rapid, sensitive, and visual detection of Apis mellifera filamentous virus
Ya Guo1, Tingting Ge1, Qiang Wang1
1Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, China.
Abstract:
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.
Insights
A new RPA-CRISPR/Cas12 platform offers rapid, sensitive detection of Apis mellifera filamentous virus (AmFV) in honey bees. This tool aids in early AmFV diagnosis, crucial for preventing widespread bee health decline.
Area of Science:
- Veterinary Entomology
- Molecular Diagnostics
- Apiculture Science
Background:
- Apis mellifera filamentous virus (AmFV) poses a significant threat to honey bee health, increasing susceptibility to other pathogens and causing mortality.
- Current detection methods, primarily PCR-based, lack the speed required for effective field interventions.
- Rapid and accurate AmFV detection is essential for managing outbreaks and protecting honey bee populations.
Purpose of the Study:
- To develop a rapid, ultrasensitive, and field-deployable detection platform for AmFV.
- To combine recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology for enhanced AmFV diagnostics.
- To validate the platform's specificity, sensitivity, and consistency with existing methods.
Main Methods:
- Development of a CRISPR RNA (crRNA1) targeting the AmFV Bro gene for specificity.
- Optimization of a combined RPA and CRISPR/Cas12a reaction for AmFV detection.
- Utilizing fluorescence-based and lateral flow dipstick methods for result visualization.
- Validation using field-collected honey bee samples and comparison with conventional PCR.
Main Results:
- The developed RPA-CRISPR/Cas12 platform achieved results within 35 minutes (20 min RPA, 15 min CRISPR/Cas12a).
- The platform demonstrated high sensitivity, detecting as few as 10 copies of the AmFV genome.
- Specificity was confirmed, with no cross-reactivity to other insect DNA viruses or uninfected bees.
- Field sample validation showed consistency with PCR, revealing prevalent latent AmFV infections.
Conclusions:
- A novel RPA-CRISPR/Cas12 platform enables rapid, specific, and sensitive detection of AmFV in both Apis mellifera and Apis cerana.
- This technology offers a promising solution for point-of-care AmFV diagnosis in the field.
- The platform's simplicity, accuracy, and cost-effectiveness can significantly aid in managing honey bee diseases.
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