Four Recombinase-Aided Amplification Assays for the Rapid Detection of Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) infection leads to serious damage to the poultry industry worldwide. Accurate and prompt diagnosis is essential for effective management of FAdV-4 outbreaks. In this study, we first designed a pair of specific primers based on the ORF19A gene of FAdV-4 strain NP for recombinase-aided amplification (RAA) and optimized the reaction conditions. The results revealed that the optimal conditions for a sensitive RAA reaction were at 39 °C for 40 min. The minimum detection amount of FAdV-4 genomes was 10−2 ng/μL using RAA. In order to facilitate the result presentation of RAA, which was visualized using a lateral flow dipstick (LFD), and the optimal reaction time of RAA-LFD was shortened to 30 min. However, the minimum detectable amount of FAdV-4 genomes was 50 ng/μL, which was lower than the sensitivity of RAA. Furthermore, to enhance the sensitivity of RAA-LFD, we designed LbaCas12a-FAdV-4-crRNA and incorporated it into RAA to develop the RAA-Cas12a-F and RAA-Cas12a-LFD methods, respectively. Based on the RAA reaction of 40 min, RAA-Cas12a-F and RAA-Cas12a-LFD only need an additional 15 min. The minimum detectable amounts of RAA-Cas12a-F and RAA-Cas12a-LFD were 10−6 ng/μL and 10−5 ng/μL, respectively. These results indicate a significant improvement in the sensitivity of two detection methods. A total of 192 fecal samples from chicks were collected from Fujian province. These samples were then detected using these four RAA rapid detection methods. The results showed that 57 positive samples were screened by both RAA-Cas12a-F and RAA-Cas12a-LFD, which was 4 more positive samples than the RAA method and 14 more positive samples than the RAA-LFD method. Overall, we developed four accurate and rapid RAA assays of FAdV-4, which is feasible and of certain value in the application of FAdV-4 in clinical practice.