Comparative Screening of Molecular Techniques for Detection of Foodborne Viruses in Pasteurized Milk and Mozzarella Cheese

Milk and dairy products may be contaminated by enteric viruses along the production chain, and their high protein and fat content hinders viral concentration and molecular detection. Internationally standardized recovery protocols have been established for leafy greens, soft fruits, and bivalve mollusks, but remain unavailable for dairy matrices. Three low-cost methods were compared for pasteurized milk, namely polyethylene glycol precipitation (PEG), Tris-Glycine-Beef Extract elution (TGBE), and a modified organic flocculation (FLO) exploiting the matrix’s own casein, and three for mozzarella cheese (PEG, TGBE, and glycine buffer–chloroform extraction, GBC). Samples were spiked with murine norovirus 1 (MNV-1) and murine hepatitis virus 3 (MHV-3) as non-enveloped and enveloped surrogates, and recovery was determined by RT-qPCR. TGBE yielded the highest mean recovery in both matrices (milk: 1.5 ± 0.6% for MNV-1 and 4.3 ± 0.6% for MHV-3; cheese: 4.7 ± 3.5% and 15.1 ± 17.5%), and its superiority over PEG for MNV-1 in milk was the only difference to reach statistical significance. PEG achieved recovery comparable to that of TGBE for MNV-1 in cheese (1.1 ± 1.5%) but with greater variability, whereas GBC recovery was negligible and the protocol requires a hazardous solvent. Organic flocculation yielded lower recovery (0.28 ± 0.34% for MNV-1) but allowed for processing of one-liter volumes, a potential alternative for large-volume screening of bulk milk. TGBE therefore appears the most practical candidate for viral recovery from dairy matrices in laboratories with limited infrastructure, although a larger number of replicates and validation with naturally contaminated samples remain necessary before routine application.