Development of a Rapid, Sensitive, and Visual Recombinase-Aided Amplification Combined with Pyrococcus furiosus Argonaute (RAA-PfAgo) Assay for Sex Identification in Swinhoe’s Pheasant (Lophura swinhoii)

Swinhoe’s pheasant (Lophura swinhoii) exhibits pronounced sexual dimorphism in adults. However, sexual differences are minimal in juveniles and subadults, making accurate early sex determination essential for captive breeding and conservation. Conventional PCR-based sexing methods require expensive equipment and are unsuitable for field or basic breeding facilities. We developed a novel recombinase-aided amplification (RAA) combined with a Pyrococcus furiosus Argonaute (PfAgo) nuclease system targeting the W-linked EE0.6 sequence (EE0.6–RAA-PfAgo) for rapid and visual sex identification in Swinhoe’s pheasant using DNA extracted from feather samples. In addition, a UCE-based RAA-PfAgo internal amplification control (UCE–RAA-PfAgo) was established to monitor DNA quality and amplification success. The optimal EE0.6-RAA reaction was performed at 39 °C for 20 min, followed by PfAgo-mediated product detection. The entire assay can be completed within 50 min, and results are visually interpretable under blue light. In our dilution experiments, the EE0.6–RAA-PfAgo assay showed a detection limit of 0.65 ng of genomic DNA, which was 10-fold lower than that of conventional PCR (6.5 ng). In a blind test of 19 field feather samples, the results were 100% concordant with conventional PCR. This study establishes a rapid, sensitive, convenient, and equipment-minimizing method for sex identification in Swinhoe’s pheasant, suitable for field applications and basic captive breeding facilities.