|Engelbrekston, Anna - RAININ INDUSTRIES|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 20, 2008
Publication Date: July 20, 2008
Citation: Tian, P., Engelbrekston, A., Mandrell, R.E. 2008. Two-log increase in sensitivity for detection of Norovirus in complex samples using porcine gastric mucin capture and immunomagnetic separation. Applied and Environmental Microbiology 74(14)4271-4276 Interpretive Summary: Human histo-blood group antigens (HBGA) have been identified previously as candidate receptors for human norovirus (NOR). HBGAs of blood group types A, H1 (related to O type), and Lewis have been identified as the major types that bind NOR. We have identified that pig stomach (gastric) mucin (PGM) also contains blood group A, H1, and Lewis (Lewis type b) HBGAs and binds more strains of NOR than do specific antibodies to NOR. NOR strains representing the I and II genogroups and present in dilute samples could be captured and concentrated by PGM-conjugated to magnetic beads, and NOR on beads were available for further processing. NOR present in a human patient fecal sample was detectable at a dilution of 1: 1,000,000 using the standard protocol RNA extraction procedure, whereas, NOR was detectable at a 1: 100,000,000 dilution due to efficient capture and concentration of NOR by PGM-beads. Moreover, NOR present in spiked complex food samples (e.g. oyster extract, strawberry, raspberry, and lettuce) also were captured by PGM, a procedure that increased the sensitivity of the real time-PCR assay by minimizing the interference by known inhibitors in food.
Technical Abstract: Human histo-blood group antigens (HBGA) have been identified previously as candidate receptors for human norovirus (NOR). Type A, type H1, Lewis HBGAs have been identified major HBGA for NOR binding. We have identified that pig stomach mucin (PGM) contains group A, type H1, and Lewis b type HBGAs and have a broader coverage than specific antibodies to bind to multiple strains of NOR. Both genotype I and II NORs strains can be recovered by PGM-conjugated magnetic beads. When NOR was serial diluted, virus can be detected at a dilution of 1: 1,000,000 by using the standard RNA extraction procedure. However, a virus dilution of 1: 100,000,000 can be concentrated and detected by using PGM-conjugated magnetic beads. When NOR viruses were spiked into food samples (oyster extract, strawberry, raspberry, and lettuce), the viruses can be captured and the RT-PCT inhibitors which often are problematic for PCR methods in general can be removed by using PGM-conjugated magnetic beads.