Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR

Biotechniques. 2000 Jul;29(1):88-93. doi: 10.2144/00291st03.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Technical Report

MeSH terms

  • Enterovirus / genetics*
  • Fluorescent Dyes
  • RNA, Viral / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Sewage / virology


  • Fluorescent Dyes
  • RNA, Viral
  • Sewage