Quantification of Enterovirus RNA in Sludge Samples Using Single Tube Real-Time RT-PCR
| Autor: | S. Monpoeho, A. Dehée, B. Mignotte, L. Schwartzbrod, V. Marechal, J.-C. Nicolas, S. Billaudel, V. Férré |
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| Jazyk: | angličtina |
| Rok vydání: | 2000 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 29, Iss 1, Pp 88-93 (2000) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/00291st03 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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