Two-log increase in sensitivity for detection of norovirus in complex samples by concentration with porcine gastric mucin conjugated to magnetic beads

Abstract

Human histo-blood group antigens (HBGA) have been identified previously as candidate receptors for human norovirus (NOR). Type A, type H1, and Lewis HBGA in humans have been identified as major HBGA for NOR binding. We have found that pig stomach (gastric) mucin (PGM) contains blood group A, H1, and Lewis b HBGA and binds to multiple strains of NOR more broadly than do specific antibodies to NOR. Both genogroup I (GGI) and GGII NOR strains were recovered by PGM-conjugated magnetic beads. A fecal sample containing GGII NOR was detected at a dilution of 1:1,000,000 by the standard RNA extraction procedure, whereas NOR in a 1:100,000,000 dilution could be concentrated by PGM-conjugated magnetic beads and NOR in spiked food samples (e.g., oyster extract, strawberry, raspberry, and lettuce) was captured by PGM, thus minimizing the reverse transcription-PCR inhibitors in food and increasing sensitivity.

FIG. 1.

Detection limit for detection of NOR by real time RT-PCR. NOR virus was diluted serially to different concentrations. Viral RNA was extracted from each dilution and measured by the real time RT-PCR method. Virus dilutions and Ct values are shown at the top.

FIG. 2.

Concentration of NOR by PGM-MB. Samples containing various amounts of NOR were concentrated by PGM-MB. I (triangle), B (filled circle), and U (square) represent input, beads recovered, and unbound, respectively. Panels A, B, C, and D represent virus loads of 10, 1, 0.1, and 0.01 RT-PCR unit, respectively. The difference in Ct values between beads recovered and input B indicates that viruses are concentrated by using PGM-MB. A 3.3-fold difference in Ct values represents a 10-fold difference in concentrations. NOR can be concentrated at least 100-fold.

FIG. 3.

Detection of NOR in spiked oyster, lettuce, and strawberry samples. A. NOR virus was spiked in PBS (diamond) or in oyster (triangle) samples at a concentration of 10 RT-PCR units and was concentrated by using PGM-MB. An equivalent amount of NOR virus was spiked into oyster samples, and virus RNA was extracted by the standard RNA extraction method (square). Un-spiked oyster samples were used as negative controls (circle). Similar Ct values for recovery of NOR from PBS and oyster samples suggest that inhibitors are removed by PGM-MB. The presence of inhibitors in oyster samples was confirmed in oyster samples where viral RNA was extracted directly (square). B. NOR virus was spiked into lettuce (diamond) or strawberry (triangle) samples at a concentration of 10 RT-PCR units and was concentrated by using PGM-MB. An equivalent amount of NOR virus was spiked into lettuce (circle) and strawberry (square), and virus RNA was extracted by the standard RNA extraction method as an input control.