# Establishment of a One-Step Rapid Visual Detection Method for Pigeon Circovirus Based on the RAA-CRISPR/Cas12a Assay

## TL;DR

This paper introduces a fast and accurate method to detect pigeon circovirus using a combination of RAA and CRISPR/Cas12a technology, enabling on-site testing within 30 minutes.

## Contribution

A one-step, closed-system visual detection method for pigeon circovirus with high sensitivity and specificity is developed.

## Key findings

- The method detects as low as 6.08 copies/µL of PiCV with high sensitivity.
- It shows no cross-reactivity with other viruses, ensuring high specificity.
- The method achieved a 92.5% coincidence rate with qPCR when testing clinical samples.

## Abstract

Pigeon circovirus is one of the important pathogens that threaten the development of the pigeon industry. Rapid and accurate detection is crucial for preventing the outbreak of this disease. In this study, we combined the efficient recombinase-aided amplification (RAA) isothermal amplification technology with the precise recognition and strong signal amplification capabilities of the CRISPR/Cas12a assay, then developed a rapid and accurate diagnostic method for pigeon circovirus. This method is easy to operate and supports on-site rapid detection, with results available within 30 min. Meanwhile, it has high specificity and shows no cross-reactions with other viruses, and can detect extremely low viral loads. More innovatively, the entire detection process is completed in a closed system, which significantly reduces the risk of aerosol contamination caused by lid-opening operations and effectively lowering the risk of false positives. This method provides an important technical means for then on-site rapid diagnosis of Pigeon circovirus and demonstrates broad prospects for clinical application.

Pigeon circovirus (PiCV) is an important pathogen that infects pigeons, which can induce multiple disorders such as immunosuppression and respiratory symptoms, posing a serious threat to the pigeon industry. In this study, we combined the RAA and CRISPR/Cas12a assay to establish a highly sensitive and accurate detection method for PiCV. This detection method amplifies the target nucleic acids through RAA; and the resultant dsDNA is specifically recognized by crRNA, the trans-cleavage activity of Cas12a is activated, which further cleaves the fluorescent reporter group to generate a fluorescent signal that can be visually observed under blue light. The method established in this study exhibited high sensitivity, with a minimum detection limit of 6.08 copies/µL. It showed no cross-reactivity with non-PiCV samples, demonstrating high specificity. When 40 clinical samples were tested by this method and quantitative polymerase chain reaction (qPCR) respectively, the coincidence rate was 92.5%, and the method developed herein achieved a higher positive detection rate. In conclusion, we successfully developed a rapid, on-site operable, one-step visual detection method for PiCV, which holds promising application prospects.