title: Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.
abstract: We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.
authors.affiliation: Laboratoire de Virologie CHU de Nantes, France.
authors.forename: S
authors.initials: S
authors.lastname: Monpoeho
authors.forename: A
authors.initials: A
authors.lastname: Dehée
authors.forename: B
authors.initials: B
authors.lastname: Mignotte
authors.forename: L
authors.initials: L
authors.lastname: Schwartzbrod
authors.forename: V
authors.initials: V
authors.lastname: Marechal
authors.forename: J C
authors.initials: JC
authors.lastname: Nicolas
authors.forename: S
authors.initials: S
authors.lastname: Billaudel
authors.forename: V
authors.initials: V
authors.lastname: Férré
mesh_terms: D004770:Enterovirus / Q000235:genetics*; D005456:Fluorescent Dyes; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012722:Sewage / Q000821:virology
chemical_list: D005456:Fluorescent Dyes; D012367:RNA, Viral; D012722:Sewage