Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
J Virol Methods. 2010 Dec;170(1-2):42-6. doi: 10.1016/j.jviromet.2010.08.017. Epub 2010 Sep 8.
Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34-60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5-5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.
A 组轮状病毒（RV-A）是全球儿童病毒性胃肠炎最常见的病原体。本研究的目的是比较两种不同的方法，从污水样本中浓缩 RV-A，并使用带有内部对照的多重定量 PCR 检测方法提高 RV-A 的检测和定量能力。将 RV-A 和内部对照病毒噬菌体 PP7 接种到废水中，然后分别使用基于超滤的吸附洗脱法或基于超速离心的方法进行浓缩。使用实时多重定量 PCR 定量纯化的 RV-A 和 PP7，并将多重检测的结果与单重检测的结果进行比较。基于超速离心的方法的平均回收率为 47%（范围：34-60%），而基于超滤的吸附洗脱法的平均回收率为 3.5%（范围：1.5-5.5%）。这些结果表明，超速离心法是从废水中回收 RV-A 的更合适方法。该方法与多重 qPCR 检测方法一起，可能适合常规实验室使用。
