Development of a semi-quantitative real-time RT-PCR for the detection of measles virus

Abstract

Real-time detection of polymerase chain reactions allows convenient detection and quantification of virus-derived nucleic acids in clinical specimens. We have developed a real-time RT-PCR assay for the detection of measles virus (MV) genomic RNA, and compared it to a well-established conventional RT-PCR assay. Based on a serial dilution of the live-attenuated MV Edmonston Zagreb vaccine, the detection limits were approximately 0.1 and 0.02 cell culture infectious dose 50% units (CCID50) per test for the conventional and TaqMan RT-PCR assays, respectively. Furthermore, tissue materials spiked with known quantities of MV were equally well detected in both assays. The TaqMan assay was linear within a range of 10(4.4) to 10(-0.6)CCID50/ml, with an intra-assay variability lower than 3% and an inter-assay variability ranging from 1.5% at 10(4.4)CCID50/ml to 8.7% at 10(-0.6)CCID50/ml. The TaqMan assay could detect representative wild-type viruses from the currently active MV clades, and could detect MV genome in clinical specimens obtained from measles patients. Finally, quantification of MV RNA in peripheral blood mononuclear cells or broncho-alveolar lavage cells from cynomolgus macaques collected at different time points after experimental infection showed a good correlation with virus isolation data. In conclusion, the TaqMan assay developed is specific, sensitive, rapid and reproducible, and can be of use for diagnostic purposes or for studies on the pathogenesis of measles.