title: An integrated cell culture and reverse transcription quantitative PCR assay for detection of infectious rotaviruses in environmental waters.
abstract: Rotaviruses exist widely in water environments and are the major cause to the gastroenteritis in children. To overcome the limitations associated with the current methods for detecting rotaviruses in environmental samples, such as long duration with the traditional cell culture-based plaque assay, inability to detect infectivity with RT-PCR-based molecular methods and lower sensitivity with ELISA tests, we developed an integrated cell culture and reverse transcription quantitative PCR (ICC-RT-qPCR) assay to detect infectious rotaviruses based on detection of viral RNA during replication in cells. The cell culturing step before qPCR allows the infectious rotaviruses to replicate and be detected because they are the only ones that can infect cells and produce RNA. The results showed that as low as 0.2 PFU/ml rotaviruses were detected by ICC-RT-qPCR after 2 days of incubation. With samples, the copy numbers of VP7 gene of rotaviruses linearly correlated (with a coefficient (R(2)) of 0.9575) with initial virus concentrations ranging from 0.2 to 200 PFU/ml. In parallel comparing tests, the ICC-RT-qPCR exhibited higher sensitivity than both the plaque assay and the RT-qPCR when applied to field samples. ICC-RT-qPCR detected infectious rotavirus in 42% (10/24) of secondary effluents, while only 21% (5/24) and 12% (3/24) of samples were positive with either the plaque counting or the RT-qPCR method, respectively. Concentrations of rotaviruses in secondary effluent samples were determined to be 1-30 PFU/l. The results demonstrated that the developed ICC-RT-qPCR method reduced test duration and improved sensitivity towards infectious rotavirus and therefore can be an effective and quantitative tool for detecting infectious rotaviruses in water environments.
authors.affiliation: Environmental Simulation and Pollution Control (ESPC) State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: April Z
authors.initials: AZ
authors.lastname: Gu
authors.forename: Wan
authors.initials: W
authors.lastname: Yang
authors.forename: Miao
authors.initials: M
authors.lastname: He
authors.forename: Xiu-Hua
authors.initials: XH
authors.lastname: Hu
authors.forename: Han-Chang
authors.initials: HC
authors.lastname: Shi
mesh_terms: D002460:Cell Line; D005618:Fresh Water / Q000821:virology*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000821:virology*; D014776:Virus Cultivation / Q000379:methods*