ABSTRACT
The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgonne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179-87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.
Subject(s)
Measles-Mumps-Rubella Vaccine/immunology , Polymerase Chain Reaction/methods , Animals , Base Sequence , Chlorocebus aethiops , DNA Primers , Measles virus/pathogenicity , Measles virus/physiology , Measles-Mumps-Rubella Vaccine/genetics , Mumps virus/pathogenicity , Mumps virus/physiology , RNA, Viral/genetics , Reproducibility of Results , Rubella virus/pathogenicity , Rubella virus/physiology , Vero Cells , Virulence , Virus ReplicationABSTRACT
Analysis of optically active compounds in complex samples is often based on chiral chromatography or capillary electrophoresis in order to separate the enantiomers. This requires a chiral reagent, when using conventional chromatography, or an expensive chiral column, or a chiral selector, when using capillary electrophoresis. The type of column, reagent, or additive depends highly on the compound to be analysed. A simple and generally applicable method is using a conventional HPLC column coupled to a CD detector. Separation of enantiomers is not required, as they can be identified by a positive or negative peak. A racemate does not produce a peak; neither does an optically inactive compound. The application of HPLC-CD for the identification of pharmacologically active compounds, such as dexamphetamine, 5-hydroxytryptophan, (-)-huperzine A, and interferon, as standards, in registered drugs, in falsifications, and in food supplements is described.
