A novel efficient pretreatment device integrated with chitosan aerogel and hydrogel composite membrane for detection of norovirus in seafood.

Norovirus (NoV) GII.4 is a major causative agent of foodborne illness, with its contamination of shellfish and other marine products posing a significant public health risk. Therefore, there is an urgent need to develop efficient on-site detection methods. However, current sample pretreatment technologies are often associated with limitations such as bulky instrumentation, high operational costs, and time-consuming procedures, which hinder the widespread adoption and practical application of on-site detection solutions. This study presents an innovative "two-step series" micro-pretreatment device (TSMPD) that utilizes membrane material pore size regulation. The primary materials employed are chitosan aerogel and nanofiber hydrogel composite membranes. The technical core of this approach involves the initial rapid interception and efficient separation of complex food sample matrices through a large-pore, high-adsorption aerogel layer, which effectively removes matrix interference. Subsequently, a nanofiber hydrogel composite membrane featuring a small-pore dense structure is utilized to achieve efficient enrichment and recovery of target virus particles. In line with the aforementioned enrichment principle, oysters were selected as representative food samples in this study, successfully facilitating the efficient enrichment and concentration of NoV GII.4 within oyster samples. Notably, virus enrichment was accomplished within 80 min, achieving a recovery rate of 25.35 %. Meanwhile, in conjunction with the RPA-CRISPR/Cas12a fluorescence detection system, a rapid and sensitive quantitative detection method for NoV GII.4 in food has been developed. The results indicate that the integrated technical solution of "enrichment - detection" established in this study can facilitate on-site rapid screening of norovirus-contaminated positive samples within 2h, achieving a detection limit of 97 copies/μL. Furthermore, this method demonstrates excellent specificity and stability. When compared to the gold standard for nucleic acid testing, the findings revealed a high degree of consistency (p>0.05), notably reducing the total duration of the entire testing process by half. This method offers several advantages including ease of operation, reduced time consumption, and low cost, thereby providing reliable technical support for on-site rapid screening of NoV GII.4 in food products.