Detection of norovirus capsid proteins in faecal and food samples by a real time immuno-PCR method

AIMS: To develop a sensitive real time immuno-polymerase chain reaction (rtI-PCR) method for detecting norovirus (NV) capsid protein in food samples. METHODS AND 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. CONCLUSION: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a rtI-PCR method to detect NV in contaminated food samples without concentration or purification of the virus.