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...
| Veröffentlicht in: | BioTechniques. - 1988. - 29(2000), 1 vom: 17. Juli, Seite 88-93 |
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| 1. Verfasser: | |
| Weitere Verfasser: | , , , , , , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2000
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Research Support, Non-U.S. Gov't Technical Report Fluorescent Dyes RNA, Viral Sewage |
| Zusammenfassung: | 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 |
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| Beschreibung: | Date Completed 09.11.2000 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 1940-9818 |