Abstract
Globally, norovirus has become the primary cause of outbreaks of acute gastroenteritis, and an increasing number of norovirus GII infections have been associated with shellfish. This highlights the urgent need to establish sensitive and rapid detection platforms for timely screening of contaminated shellfish to reduce the risk of virus transmission. To address this challenge, we developed a novel detection method combining multienzyme isothermal rapid amplification (MIRA) with qPCR, referred to as MIRA-qPCR, specifically targeting norovirus GII. It exhibited robust specificity, demonstrating no cross-reactivity with sapovirus, rotavirus, hepatitis A virus, Escherichia coli, Listeria monocytogenes, or Vibrio parahaemolyticus, and exhibited high sensitivity, detecting as low as 1.62 copies/μL for recombinant plasmid standards. Furthermore, MIRA-qPCR showed good linearity in the 1.62 × 10(1) to 1.62 × 10(7) copies/μL range, with an R(2) > 0.90. MIRA-qPCR and qPCR assays were performed on 125 fresh shellfish samples; there was good consistency in the detection results, and the Kappa value was 0.90 (p < 0.001). The sensitivity and specificity of the MIRA-qPCR detection were 100.00% and 97.25%, respectively. The MIRA-qPCR technique provides a viable alternative for the rapid screening of norovirus GII-contaminated shellfish to guarantee food safety.