Application of a receptor-binding capture quantitative reverse transcription-pcr assay to concentrate human norovirus from sewage and to study the distribution and stability of the virus

Abstract

Water is an important route for human norovirus (HuNoV) transmission. Usingmagnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture andmagnetic sequestration (RBCMS)method and compared it to the existing negatively chargedmembrane absorption/elution (NCMAE)method for con-centrating HuNoV fromsewage efuent. RBCMS required 6-fold-less sample volume than the NCMAEmethod and also resulted in a signicantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoVmeasured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV weremeasured as average CT values of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specicity of qRT-PCR was conrmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal fromRBCMS-concentrated HuNoV treated with RNase I indicated that it was fromencap-sidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal fromNCMAE-concentrated HuNoV was not protected fromRNase I and, likely, degradation. Both GI and GII HuNoV were detected fromsewage efuent samples collected between April and July with average concentrations of 7.8×103 genomic copies per liter (gc/liter) and 4.3×104 gc/liter, respectively. No GI and<2%GII HuNoV were detected in sewage samples stored at roomtemperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage. © 2012, American Society for Microbiology.