Introduction: Recovery of human noroviruses from food and water using Porcine Gastric Mucin (PGM)-functionalized superparamagnetic beads has recently been described. However, the assay has not been directly compared to immune-magnetic separation and the lower limit of detection is not known.
Purpose: To assess the applicability of this method, capture human noroviruses by PGM- and monoclonal antibody (mAB)-coated magnetic beads was compared in the context of elution buffers that are commonly used in food virology.
Methods: Amine- and tosyl-functionalized superparamagnetic beads were coated with PGM and mAB (NV3901), respectively, or left uncoated to determine non-specific binding of virus to beads. The beads were re-suspended in glycine (pH 7.0 and 9.5), 1M NaCl (pH 7.4), or citrate (pH 3.6) buffers containing 10-fold serial dilutions of Norwalk (GI.1) virus. After attachment and washing, viral RNA was detected by realtime RT-qPCR. The lower detection limit (LDL) for each bead/buffer combination was reported by expressing the lowest viral inoculum yielding positive RT-PCR results.
Results: For PGM-coated beads, LDLs were similar in the context of all buffers tested; 3.3, 3.3, 3.6 and 3.7 log genome copy numbers for glycine (pH 7.0), citrate, glycine (pH 9.5) and 1M NaCl buffers, respectively. With the mAB-coated beads, LDLs were generally higher; 4.5, 4.3 and 4.9 log genome copies for glycine (pH 7.0), citrate and 1M NaCl buffers, respectively. The mAB-coated beads in glycine buffer (pH 9.5) yielded the lowest LDL of the group (3.5 log genome copy numbers). Non-specific binding was problematic, especially for uncoated tosyl beads in 1M NaCl buffer and for all cases when virus input approached 5 log genome copies or more.
Significance: PGM-coated beads were the most versatile, out-performing the mAB-coated beads in nearly all buffer matrices. However, because LDLs were higher than desired, further optimization is needed before implementation in a foodborne outbreak investigation.