Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2005
Publication Date: 2/5/2006
Citation: Tian, P., Mandrell, R.E. 2006. Detection of norovirus capsid in fecal and food samples by a real time immuno-PCR method. Journal of Applied Microbiology. 100:564-574.
Interpretive Summary: We have developed a sensitive real time immuno-PCR (rtI-PCR) method for the detection of Norwalk-like (NoV) viruses in food samples. NoV or recombinant Norwalk viral-like particles (rNVLPs) were used as antigens for the rtI-PCR assay. The sensitivity of rtI-PCR was >1000 fold higher than the standard ELISA. The sensitivity of rtI-PCR was also better than RT-PCR in detection of NoV virus in food samples and stool samples.
Technical Abstract: Noroviruses (NoV), previously known as “Norwalk-like viruses”, cause outbreaks of gastroenteritis and are spread frequently through contaminated food or water. Enzyme-Linked Immunosorbent Assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) are the most common diagnostic methods for virus detection. ELISA is used infrequently for detection of Norwalk-like viruses in food samples because of low sensitivity. As numbers of contaminating viruses present in food are relatively low and inhibitors are present, complex purification and concentration methods must be used prior to RT-PCR In our newly developed system, the viral antigens were captured by two polyclonal antisera (gunea pig and rabbit) against rNVLPs. Biotin-conjugated anti-rabbit antibodies, avidin, and biotin-conjugated DNA reporter were added to the system to convert the protein signals into DNA signals. The reporter DNA was then amplified by addition of primers and PCR. The rtI-PCR method developed could detect as low as 0.1 pg recombinant Norwalk like viral capsid proteins. In other methods with food samples, many of which contain strong PCR inhibitors, the inhibition effect has been removed by diluting the sample 1000-fold, or by other purification steps. Our results suggest that the PCR inhibitors present in the food samples tested have minimal effect on antigen capture and could be removed by multiple wash steps during the rtI-PCR procedure. Therefore, the rtI-PCR method provides an improved method for detection of Norwalk virus contaminating food samples.