# A RPA-CRISPR/Cas12a-Powered Catalytic Hairpin Assembly Fluorescence Biosensor for Duck Plague Virus Virulent Strain Detection

## TL;DR

A new biosensor using RPA, CRISPR/Cas12a, and CHA was developed to quickly and accurately detect a dangerous duck virus.

## Contribution

A novel fluorescence biosensor combining RPA, CRISPR/Cas12a, and CHA for enhanced detection of virulent duck plague virus strains.

## Key findings

- The biosensor detected DPV with a detection limit of 0.02 fg/μL in 40 minutes.
- It showed five-fold better sensitivity than traditional RPA-CRISPR/Cas12a biosensors.
- The biosensor results were consistent with clinical and PCR diagnosis methods.

## Abstract

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings.