Detection of norovirus capsid proteins in faecal and food samples by a real time immuno-PCR method.
OBJECTIVE
To develop a sensitive real time immuno-polymerase chain reaction (rtI-PCR) method for detecting norovirus (NV) capsid protein in food samples.


RESULTS
The viral antigens were captured by two polyclonal antisera against recombinant Norwalk viral-like particles (rNVLPs). Biotin-conjugated antibodies, avidin and biotin-conjugated DNA reporter were used to convert the protein signals into DNA signals. The reporter DNA was then amplified by addition of primers and PCR. A real time PCR method was used in order to perform a quantitative post-PCR analysis. One hundred rNVLPs (10 fg) and a NV sample containing 660 rNVLPs equivalent particle units (66 fg) could be detected by this method.


CONCLUSIONS
The PCR inhibitors present in the food samples had minimal effect on antigen capture and were removed by multiple wash steps during the rtI-PCR procedure. The sensitivity of rtI-PCR was >1000-fold higher than the standard enzyme-linked immunosorbent assay and approximately 10 times higher than reverse transcription PCR in detection of NV capsid protein in stool and food samples.


CONCLUSIONS
This is the first report of a rtI-PCR method to detect NV in contaminated food samples without concentration or purification of the virus.
United States Department of Agriculture, Agricultural Research Service, Produce Safety and Microbiology Research Unit, Western Regional Research Center, Albany, CA 94710-1105, USA. ptian@pw.usda.gov
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Mandrell
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D036022:Capsid Proteins / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005243:Feces / Q000737:chemistry* / Q000821:virology; D005504:Food Analysis / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D031985:Fragaria / Q000737:chemistry / Q000276:immunology; D029322:Norovirus / Q000737:chemistry* / Q000276:immunology; D010125:Ostreidae / Q000737:chemistry / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012758:Shellfish / Q000032:analysis
D000956:Antigens, Viral; D036022:Capsid Proteins; C121877:Norwalk virus capsid; D012367:RNA, Viral; D011994:Recombinant Proteins