Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration

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. (C) 2010 Elsevier B.V. All rights reserved.