A dual enhancement strategy for ultrasensitive detection of norovirus based on catalytic hairpin assembly–assisted CRISPR/Cas12a sensing system

Noroviruses are a leading cause of food-borne illnesses, responsible for over 50% of global gastroenteritis outbreaks. Whereas reverse transcriptase PCR (RT-PCR) and other nucleic acid amplification methods are crucial for norovirus detection, their reliance on specialized equipment highlights the urgent need for more accessible detection methods. Herein, we propose an isothermal cascade signal amplification assay that integrates catalytic hairpin assembly (CHA) and cluster regularly interspaced short palindromic repeats (CRISPR)/Cas12a for rapid and accurate detection of norovirus in food samples. By incorporating the advantages of CHA and CRISPR/Cas12a, the dual signal enhancement sensing strategy can achieve high sensitivity low to 14 fM within 70 min, and good specificity in adenovirus, human enterovirus, rotavirus, and other interfering agents. The proposed dual enhancement strategy for norovirus detection has satisfactory accuracy and acceptable recoveries in milk samples compared with RT-PCR assay, and holds promise for improving food safety monitoring, particularly in dairy products.