Systematic evaluation of the micro-volume PEG-enrichment pretreatment for norovirus detection across seven commercially available bivalve species.

Norovirus (NoV) is a major cause of foodborne gastroenteritis worldwide. Filter-feeding bivalves can accumulate NoV from aquatic environments and are important vehicles of foodborne transmission. Our group previously developed a micro-volume PEG-enrichment pretreatment (MEP) method for viruses in bivalves based on ISO 15216; however, its applicability in various bivalve species remains unclear. Therefore, this study systematically compared the MEP and ISO methods for NoV detection in seven commercial bivalve species: Crassostrea gigas, Mytilus galloprovincialis, Meretrix meretrix, Argopecten irradians, Tegillarca granosa, Ruditapes philippinarum and Sinonovacula constricta. HNoV recovery ranged from 2.15% to 4.79% using ISO and increased to 13.96%-56.69% using MEP, with significant interspecies differences (p < 0.05). Recovery was highest in Mytilus galloprovincialis (56.69 ± 1.92%), followed by Sinonovacula constricta (24.81 ± 1.63%) and Meretrix meretrix (23.31 ± 1.89%), and lowest in Tegillarca granosa (13.96 ± 1.22%). Virus concentrations obtained using the MEP method were 4.36- to 22.23-fold those obtained using the ISO method. RT-qPCR amplification efficiencies ranged from 70.66% to 104.35% for the ISO and from 65.67% to 99.46% for MEP, with significant interspecies differences (p < 0.05). In commercial bivalve samples analyzed using the MEP, process recovery and RT-qPCR amplification efficiency ranged from 7.67% to 51.79% and from 50.25% to 126.31%, respectively. Although virus recovery using the MEP varied among bivalve species, it was consistently higher than that obtained using the ISO method. Overall, the MEP method provides a high-performance and broadly applicable pretreatment strategy for NoV detection in bivalves, with potential for routine monitoring and surveillance.