Localized CRISPR/Cas13a powered DNA walker for sensitive and high-throughput detection of norovirus

Abstract

The development of sensitive and high-throughput methods for detecting foodborne viruses is crucial for disease prevention and public health protection. In this study, we present a novel localized Cas13a-based DNA walker (LCas13a-DNA walker) for the ultrasensitive, stable, and rapid detection of norovirus (NoV). When the DNA walker was confined in AuNPs, the spatial confinement effect improved the local concentration of reaction substrates, accelerated the reaction speed, and enhanced the sensitivity of the DNA walker. Besides, an original design of uracil-rich hairpin (UH)-modified AuNPs as the walking track significantly improves the stability of the detection system. Meanwhile, employing CRISPR/Cas13a as the driving force streamlines viral RNA recognition and substantially reduces the reaction time down to 30 minutes by eliminating the reverse transcription step. Additionally, a biomimetic array, formed by photonic crystals (PCs), enabled high-throughput signal acquisition with a microplate reader, and concurrently amplified the fluorescence signal. The proposed assay realized ultra-sensitivity of NoV with a detection limit as low as 4.1 pM and a wide linear range from 10 pM to 5 nM. Due to the advantages of high sensitivity, high-throughput, stability, and rapid analysis, this proposed method provides a potential strategy for point-of-care detection of pathogenic viruses in food safety monitoring and disease diagnosis.