title: Development and application of a quantitative RT-PCR potency assay for a pentavalent rotavirus vaccine (RotaTeq).
abstract: 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 24h. 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.
authors.affiliation: Bioprocess & Bioanalytical Research, Merck Research Laboratories, Merck & Co., Inc., WP75-300, West Point, PA 19486, USA. todd_ranheim@merck.com
authors.forename: Todd
authors.initials: T
authors.lastname: Ranheim
authors.forename: Pamela K
authors.initials: PK
authors.lastname: Mathis
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Joelsson
authors.forename: Mary Ellen
authors.initials: ME
authors.lastname: Smith
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Campbell
authors.forename: Gwendolyn
authors.initials: G
authors.lastname: Lucas
authors.forename: Scott
authors.initials: S
authors.lastname: Barmat
authors.forename: Erin
authors.initials: E
authors.lastname: Melissen
authors.forename: Robert
authors.initials: R
authors.lastname: Benz
authors.forename: John A
authors.initials: JA
authors.lastname: Lewis
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Timothy
authors.initials: T
authors.lastname: Schofield
authors.forename: Robert D
authors.initials: RD
authors.lastname: Sitrin
authors.forename: John P
authors.initials: JP
authors.lastname: Hennessey
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000502:physiology; D022243:Rotavirus Vaccines / Q000235:genetics; D014613:Vaccines, Attenuated / Q000235:genetics; D014709:Vero Cells; D010948:Viral Plaque Assay; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; C492535:RotaTeq; D022243:Rotavirus Vaccines; D014613:Vaccines, Attenuated