Development of a CRISPR/RspCas13d-based on-site rapid detection system for GII Norovirus.

Noroviruses (NoVs) are major cause of acute viral gastroenteritis and a serious public health concern. Current detection methods are limited in rapidity, equipment requirements, or sensitivity. In this study, we developed a rapid, sensitive, and specific detection assay for GII NoV by combining RT-RAA, T7 transcription, and the RspCas13d system. The RspCas13d protein was expressed and purified. RT-RAA primers and crRNA were designed against the conserved region of GII Nov. The assay was optimized and evaluated for specificity, sensitivity, and clinical performance. Results showed that the RT-RAA-RspCas13d method exhibited high specificity without cross-reactivity to other common enteric viruses. The limit of detection was 5 copies/μL. In clinical fecal samples, the assay showed high consistency with RT-qPCR. This method is rapid, simple, sensitive, and specific, providing a reliable tool for the rapid on-site detection of GII NoV.