Sensitivity-enhanced hydrogel digital RT-LAMP with in situ enrichment and interfacial reaction for norovirus quantification in food and water

Abstract

Low levels of human norovirus (HuNoV) in food and environment present challenges for nucleic acid detection. This study reported an evaporation-enhanced hydrogel digital reverse transcription loop-mediated isothermal amplification (HD RT-LAMP) with interfacial enzymatic reaction for sensitive HuNoV quantification in food and water. By drying samples on a chamber array chip, HuNoV particles were enriched in situ. The interfacial amplification of nucleic acid at the hydrogel-chip interface was triggered after coating HD RT-LAMP system. Nanoconfined spaces in hydrogels provided a simple and rapid "digital format" to quantify single virus within 15 min. Through in situ evaporation for enrichment, the sensitivity level was increased by 20 times. The universality of the sensitivity-enhanced assay was also verified using other bacteria and virus. Furthermore, a deep learning model and smartphone app were developed for automatic amplicon analysis. Multiple actual samples, including 3 lake waters, strawberry, tap water and drinking water, were in situ enriched and detected for norovirus quantification using the chamber arrays. Therefore, the sensitivity-enhanced HD RT-LAMP is an efficient assay for testing biological hazards in food safety monitoring and environmental surveillance.

Keywords: Food and water; Hydrogel digital RT-LAMP; In situ enrichment; Interfacial enzymatic reaction; Norovirus.