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.
Fumian
Tulio M
TM
Laboratory of Comparative and Environmental Virology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. tuliomf@ioc.fiocruz.br
Leite
José Paulo G
JP
Castello
Alejandro A
AA
Gaggero
Aldo
A
Caillou
Maria Susana L de
MS
Miagostovich
Marize P
MP
D000327:Adsorption; D005759:Gastroenteritis / Q000821:virology; D006801:Humans; D017909:Levivirus; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012722:Sewage / Q000821:virology*; D014461:Ultracentrifugation / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods; D014871:Water Microbiology
D012367:RNA, Viral; D012722:Sewage