Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq)
| Autor: | Erin Melissen, Todd Ranheim, Gwendolyn Lucas, Jie Chen, John A. Lewis, Robert Benz, Mary Ellen Smith, Daniel B. Joelsson, Pamela K. Mathis, Robert D. Sitrin, Scott L. Barmat, John P. Hennessey, Timothy L. Schofield, Kathryn M. Campbell |
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| Rok vydání: | 2005 |
| Předmět: |
Virus quantification
Viral Plaque Assay Rotavirus Reverse Transcriptase Polymerase Chain Reaction Rotavirus Vaccines Biology medicine.disease_cause Vaccines Attenuated Virus Replication Virology Molecular biology Virus Real-time polymerase chain reaction Viral replication Reassortant Viruses Chlorocebus aethiops medicine Potency Animals RNA Viral Vero Cells |
| Zdroj: | Journal of virological methods. 131(2) |
| ISSN: | 0166-0934 |
| Popis: | A sensitive and reproducible method to determine the in vitro infectious potency of a pentavalent reassortant rotavirus vaccine (RotaTeq®) has been developed as an alternative to classical potency assays. Potency was determined based on cell-based viral replication followed by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In the assay, confluent Vero cell monolayers in 96-well plates were inoculated with serial dilutions of test samples, a pentavalent reassortant rotavirus reference standard and assay controls, followed by incubation for 24 h. The cells were lysed with a Triton X-100 solution and the lysates assayed by RT-QPCR to quantitate viral nucleic acid produced during replication. The RT-QPCR utilizes primer/probe sets specific to each virus reassortant and the potencies of each sample were determined relative to the reference standard. This assay, hereafter referred to as the Multivalent QPCR-Based Potency Assay (M-QPA), permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. In addition, the assay was demonstrated to be concordant with a traditional method (plaque assay) for the quantitation of infectious virus particles. It is anticipated that assays of this type will become a valuable tool in the assignment of potency values and in the monitoring of stability of live virus vaccines. |
| Databáze: | OpenAIRE |
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