CRISPR-Cas13a Coupled with a Capsid Integrity Assay for Detecting Norovirus in Food

Axel Ossio, Universidad Autonoma de Nuevo Leon

Axel Ossio. - Laboratorio de Bioquímica y Genética de Microorganismos, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Angel Merino-Mascorro. - Laboratorio de Bioquímica y Genética de Microorganismos, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Juan S. Leon. - Hubert Department of Global Health, Rollins School of Public Health, Emory University. Norma Heredia. -Laboratorio de Bioquímica y Genética de Microorganismos, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Santos García. -Laboratorio de Bioquímica y Genética de Microorganismos, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León.

Advances in diagnostics and detection

Standard detection methods for human norovirus, such as RT-qPCR, identify viral RNA but cannot differentiate infectious from non-infectious particles, complicating food safety decisions. Additionally, RT-qPCR assays require costly equipment and reagents, limiting access to testing. To address both issues, we developed CRISPR FRESH (Foodborne RNA-virus Enzymatic Sensing for High-throughput), a CRISPR-Cas13a-based detection method combined with an RNase-capsid integrity assay to selectively detect intact, potentially infectious norovirus particles on fresh produce. Using murine norovirus (MNV-1) as a surrogate, CRISPR FRESH achieved a limit of detection of 3.59 log10 gc/25 g sample (5.06 × 10^1 gc/rx) on lettuce and blueberry samples. The assay showed no cross-reactivity with synthetic DNA targets of hepatitis A virus, human norovirus GI/GII, or rotavirus. Compared to RT-qPCR, CRISPR FRESH demonstrated enhanced sensitivity for detecting low viral copy numbers after RNase pretreatment. While RT-qPCR remains the method of choice for quantification, CRISPR FRESH provides a simpler, faster, and more accessible alternative for screening potentially infectious norovirus in food. This study underscores the utility of CRISPR-based diagnostics to improve food safety monitoring by targeting infectivity, aiding more informed risk assessments and management for the food industry.