Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California, USA.
Appl Environ Microbiol. 2012 Jan;78(2):429-36. doi: 10.1128/AEM.06875-11. Epub 2011 Nov 18.
Water is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), 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 required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (C(T)) 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 were measured as average C(T) values 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 by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that 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 and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 10(3) genomic copies per liter (gc/liter) and 4.3 × 10(4) gc/liter, respectively. No GI and <2% GII HuNoV were detected in sewage samples stored at room temperature 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.
水是人类诺如病毒（HuNoV）传播的重要途径。我们使用与血型样抗原（HuNoV 受体）偶联的磁珠，开发了一种简单快速的受体结合捕获和磁分离（RBCMS）方法，并将其与现有的带负电荷的膜吸附/洗脱（NCMAE）方法进行比较，以浓缩污水中的 HuNoV。与 NCMAE 方法相比，RBCMS 方法所需的样本体积少 6 倍，并且 HuNoV 的产量也显著更高。定量逆转录-PCR（qRT-PCR）测量的基因型 I（GI）HuNoV 的 NCMAE 和 RBCMS 浓度导致平均阈值循环（C（T））值分别为 34.68（8.68 拷贝，252 倍浓缩）和 34.07（13.05 拷贝，477 倍浓缩）；基因型 II（GII）HuNoV 的 NCMAE 和 RBCMS 浓度分别测量为平均 C（T）值 33.32（24.7 拷贝，239 倍浓缩）和 32.38（46.9 拷贝，333 倍浓缩）。qRT-PCR 的特异性通过传统 RT-PCR 和 RNase I 保护测定得到证实。用 RNase I 处理的 RBCMS 浓缩的 HuNoV 的 qRT-PCR 信号表明它来自包膜 RNA，可能是有活力的病毒。相比之下，用 NCMAE 浓缩的 HuNoV 的 qRT-PCR 信号不受 RNase I 的保护，可能会降解。4 月至 7 月采集的污水样本中均检测到 GI 和 GII HuNoV，平均浓度分别为 7.8×10^3 基因组拷贝/升（gc/l）和 4.3×10^4 gc/l。在室温下储存 4 周的污水样本中未检测到 GI 和 <2%的 GII HuNoV。我们得出结论，与 NCMAE 相比，RBCMS 方法需要更少的样本量，具有更好的回收率和灵敏度，并且在污水中检测 HuNoV 更快。
