Establishment and evaluation of the detection method for human adenovirus and its subtypes based on RAA-LFD technology

Abstract

Abstract: In this research, we developed a rapid assay for detecting human adenovirus (HAdV) and its prevalent subtypes (including HAdV3, HAdV4, HAdV7, HAdV14, HAdV11, and HAdV55) based on recombinase-mediated isothermal amplification combined with lateral flow chromatography (RAA-LFD). We designed specific primers and probes targeting conserved regions of the Hexon genes of HAdV and its common subtypes, and established the recombinase-aided amplification (RAA) reaction system. The optimal primer and probe combinations were identified utilizing the real-time fluorescence RAA. Subsequently, the RAA-lateral flow dipstick (RAA-LFD) reaction system was developed and optimized for reaction temperature. At the same time, the sensitivity and specificity of the method were evaluated, and 50 clinical samples were analyzed. The optimal amplification temperature for the RAA-LFD assay was determined to be 37°C, with a detection limit of 10-100 copies/μL, high specificity, and no cross-reactivity with other pathogens. Following processing of 50 clinical respiratory specimens using our laboratory's proprietary trehalose ester-based extraction-free reagent,, the RAA-LFD assay was performed and compared with conventional qPCR, showing complete agreement between the two methods (Kappa value of 1). In conclusion, this study established a simple, rapid, visual, sensitive, and specific method for the detection of HAdV and its common subtypes.