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
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 9, 2011
Publication Date: November 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.
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.