NOAA National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412-9110, USA.
J Virol Methods. 2011 Mar;172(1-2):38-45. doi: 10.1016/j.jviromet.2010.12.011. Epub 2010 Dec 24.
Norovirus is associated commonly with human sewage and is responsible for numerous cases of waterborne and foodborne gastroenteritis every year. Assays using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) have been developed for norovirus, however, accurate detection and quantitation remain problematic owing to a lack of suitable positive controls. To improve enumeration of norovirus genomes from water, a synthetic norovirus genogroup II quantitation standard and competitive internal positive control were developed. The quantitation standard demonstrates identical amplification efficiency as wildtype norovirus and can be used as a viral surrogate in labs with restricted access to norovirus. The internal control quantifies sample inhibition, allowing for accurate quantitation of norovirus from complex environmental samples. Seawater samples spiked with sewage or bird guano were evaluated using the norovirus assay as part of a methods comparison study. Inhibition was detected in nine of 36 (25%) samples, two of which proved to be positive upon re-analysis. Results support the specificity of this assay for human-source (sewage) fecal contamination. Overall, use of this quantitation standard and internal control signify a great advance over traditional positive controls and suggest that molecular techniques for viral analysis could become standardized for routine water quality monitoring.
诺如病毒通常与人类污水有关，每年都会导致许多水传播和食源性肠胃炎病例。已经开发出用于检测诺如病毒的逆转录定量聚合酶链反应 (RT-qPCR) 检测方法，但是由于缺乏合适的阳性对照，准确的检测和定量仍然存在问题。为了提高水中诺如病毒基因组的计数，开发了一种合成的诺如病毒基因 II 定量标准和竞争性内部阳性对照。定量标准与野生型诺如病毒具有相同的扩增效率，可以作为实验室中对诺如病毒有限制访问的病毒替代物。内部对照可量化样品抑制作用，从而可以准确地从复杂的环境样品中定量诺如病毒。作为方法比较研究的一部分，用诺如病毒检测方法评估了添加污水或鸟粪的海水样本。在 36 个样本中有 9 个（25%）检测到抑制，其中两个在重新分析时被证明呈阳性。结果支持该检测方法对人类来源（污水）粪便污染的特异性。总的来说，这种定量标准和内部对照的使用标志着比传统阳性对照有了重大进展，并表明病毒分析的分子技术可能成为常规水质监测的标准化方法。
