基于RAA-LFD技术的草鱼呼肠孤病毒与嗜水气单胞菌双重检测方法研究OA

To achieve rapid and accurate detection of major pathogens in grass carp(Ctenopharyngodon idella),a duplex detection method based on recombinase-aided amplification combined with lateral flow dipstick(RAA-LFD)was developed for Grass carp reovirus and Aeromonas hydrophila.Specific primers and probes targeting the vp2 gene of GCRV and the gyrB gene of A.hydrophila were designed to construct the duplex RAA-LFD system.The reaction conditions,sensitivity,specificity,and clinical applicability were systematically evaluated.The results showed that the amplification could be completed within 15 min at 37℃,and the results were visually interpreted using test strips.The RAA-LFD assay achieved a detection limit of 1 ng/μL for GCRV and 100 pg/μL for A.hy-drophila,compared to 10 pg/μL for both targets by qPCR.No cross-reactivity was observed with other common aquatic pathogens,including Vibrio parahaemolyticus,Edwardsiella tarda,Staphylococcus aureus,and koi herpes-virus.Clinical sample testing demonstrated high consistency between RAA-LFD and qPCR,with Kappa values of 0.88 and 0.91 for GCRV and A.hydrophila,respectively.In conclusion,the developed RAA-LFD method is rapid,simple,and specific,providing a practical tool for on-site detection of multiple pathogens in grass carp aq-uaculture.