Quantification of Enterovirus RNA in Sludge Samples Using Single Tube Real-Time RT-PCR

Abstract

We have developed a quantitative RTPCR 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 PrismTM 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 × 107). 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.