RAA-CRISPR/Cas12aアッセイに基づくピジョンサーコウイルスを対象としたワンステップ迅速視覚検出法の確立

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.