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United States Department of Agriculture

Agricultural Research Service

Research Project: Biology and Control of Human Pathogens on Fresh Produce

Location: Produce Safety and Microbiology Research

Title: Detection of human Norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor binding based capture and magnetic sequestration(RBCMS) method

Authors
item Pan, Liangwen -
item Zhang, Qigang -
item Li, Xiang -
item Tian, Peng

Submitted to: Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 28, 2011
Publication Date: January 11, 2012
Citation: Pan, L., Zhang, Q., Li, X., Tian, P. 2012. Detection of human Norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor binding based capture and magnetic sequestration(RBCMS) method. Food Microbiology. 30:420-426.

Interpretive Summary: Contaminated produce related norovirus (NoV) outbreak is a major public health concern. The development of a simple assay for concentrating and detecting NoV contamination in fresh produce that can be performed in a single day would be of great benefit to the producers and regulators of produce production. In this study, we develop a simple assay to concentrate NoV from various food samples that can be performed within a few hours. We identified buffers that significantly improve the release of virus from contaminated produce surfaces. We determined that the PGM-MB viral concentration method is both faster to perform and produced significantly better virus yields than the same-day PEG-precipitation concentration method. We also found that the PGM-MB viral concentration method even exceeds the virus yield of the overnight PEG-precipitation concentration method. Overall, the PGM-MB method takes significantly less time than current PEG precipitation methods, recovers a higher yield of NoV, and hence exhibits higher sensitivity.

Technical Abstract: Contaminated produce related norovirus (NoV) outbreak is a major public health concern. The establishment of a simple assay for concentrating and detecting NoV contamination in fresh produce that can be performed in a single day would be of great benefit to the producers and regulators of produce production. In this study, we tested different buffer systems for recovering human NoV from inoculated cherry tomatoes, blueberries and fresh vegetable salad with dressing, and tested different approaches for concentrating the virus. NoV could be recovered from inoculated fresh vegetable salad with dressing with neither citrate-buffered saline (CBS) nor water wash. Washing inoculated fresh vegetable salad with dressing with glycine buffer resulted in a significant improvement of virus recovery (35.57 ±0.26 Cts) relative to phosphate-buffered saline (PBS, 39.01 ±0.55 Cts). There was no difference in virus recovery with either water wash (35.84 ±0.23 Cts) or glycine wash (35.90 ±0.06 Cts) of inoculated tomatoes and blueberries. Viral RNA extraction methods were also compared. A significant improvement in post-extraction RNA yield was achieved by sequentially heat-releasing and column-extracting over either technique alone. The viral recovery of the PGM-MB method was significantly higher than the same-day PEG-precipitation method (90 min. precipitation) for all inoculated produce tested. The virus recovery of the PGM-MB method was also higher than overnight PEG precipitation method (2 days protocol). The average recovery rate for inoculated viruses by overnight PEG precipitation method was 4.33 ±1.79, 5.90 ±1.39, and 5.96 ±2.91% for blueberries, cherry tomatoes, and fresh vegetable salad with dressing, respectively. The average recovery rate by PGM-MB method was 8.72 ±3.66, 7.94 ±3.13, and 9.60 ±3.53% for blueberries, cherry tomatoes, and fresh vegetable salad with dressing, respectively. RNAse protection assay suggests that the viral genome was protected from RNA attack by remaining within the viral capsid which binds to PGM-MB. As the PGM-MB method requires both intact viral capsid protein to bind to HBGA receptors and the presence of viral genome to be amplified, the signal detected is more biologically relevant to virus infectivity. Overall, the PGM-MB method takes significantly less time than current PEG precipitation methods, and recovers a higher yield of NoV from various food samples and hence exhibits higher sensitivity.

Last Modified: 4/18/2014
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