Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR.
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.
Monpoeho
S
S
Laboratoire de Virologie CHU de Nantes, France.
Dehée
A
A
Mignotte
B
B
Schwartzbrod
L
L
Marechal
V
V
Nicolas
J C
JC
Billaudel
S
S
Férré
V
V
D004770:Enterovirus / Q000235:genetics*; D005456:Fluorescent Dyes; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012722:Sewage / Q000821:virology
D005456:Fluorescent Dyes; D012367:RNA, Viral; D012722:Sewage