CRISPR-based assays for point of need detection and subtyping of influenza

Abstract

The high disease burden of influenza virus poses a significant threat to human health and requires better methods to rapidly detect its many circulating species, subtypes, and variants. No current diagnostic technology meets the combined critical needs for a rapid, sensitive, specific, and cost-effective method for point-of-need (PON) influenza detection and discrimination with minimal equipment requirements. Here, we introduce such a method using SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a CRISPR-based RNA detection platform. We develop and validate four SHINE assays for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). These optimized assays achieve 100% concordance with reverse-transcriptase real-time polymerase chain reaction (RT-qPCR) when tested on clinical samples. We also created duplex Cas12/Cas13 SHINE assays to simultaneously detect two targets and demonstrate its use in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as to detect influenza A and human RNAse P, as a built-in internal control. Our assays have the potential to expand influenza detection outside of clinical laboratories in order to enhance influenza diagnosis and surveillance.