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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: Application of a receptor-binding-capture qRTPCR assay to concentrate human norovirus from sewage and to study the distribution and stability of the virus)

Author
item Tian, Peng
item Yang, David
item Pan, Liangwen
item Mandrell, Robert

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2011
Publication Date: 11/18/2011
Citation: Tian, P., Yang, D., Pan, L., Mandrell, R.E. 2012. Application of a receptor-binding-capture qRTPCR assay to concentrate human norovirus from sewage and to study the distribution and stability of the virus. Applied and Environmental Microbiology. 78(2):429-36 doi:10.1128/AEM.06875-11.

Interpretive Summary: Human noroviruses (HuNoVs) are major agents of epidemic gastroenteritis, and water is one of the most important routes of their transmission. We developed a simple and rapid method to concentrate HuNoV from small volumes of sewage by using magnetic beads conjugated with virus-binding blood group antigens (PGM-MB method), which is then detected with quantitative real-time RT-PCR (qRT-PCR). We found that it was necessary to remove large particulates from sewage samples prior to concentration, which may be achieved by low-speed centrifugation and pelleting, or pre-filtration with a coarse filter. We were able to achieve a higher yield of recovery with the PGM-MB method using 40 ml sewage samples than the commonly-used negative-charged membrane absorption/elution (NCMAE) method using 250 ml sewage samples. The average concentrating power of the PGM-MB method was also much better than that of the NCMAE method. Stability of HuNoV in sewage water was also investigated. The viruses were more stable at 4° C than at room temperature. In addition, HuNoV could be detected in treated sewage water during non-epidemic season (April through July). Overall, the PGM-MB method takes significantly less time and sample size than the NCMAE method, with a better recovery of HuNoV and improved sensitivity.

Technical Abstract: Human noroviruses (HuNoVs) are major agents of gastroenteritis and water is an important route of transmission. Using magnetic beads conjugated with blood group-like antigens previously reported as receptors for HuNoV, we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method, and compared it to the existing negatively-charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS concentration of dilute HuNoV required 6-fold less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE andRBCMS concentration of genogroup I (GI) HuNoV measured by qRT-PCR resulted in average Ct values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; NCMAE and RBCMS concentration of genogroup II (GII) HuNoV was measured as average Ct of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed with RT-PCR with region D primers for HuNoV in an RNase I protection assay. The qRT-PCR signal from RBCMS concentrated HuNoV treated with RNaseI indicated it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE concentrated HuNoV was not protected from RNase I activity. Both GI and GII HuNoV were detectable from sewage effluent samples collected between April and July with average concentrations of 7.8x103 genomic copies per liter (gc/l) and 4.3x104 gc/l, respectively. No GI and < 2% GII HuNoV were detectable in sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage.

Last Modified: 8/24/2016
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