Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

•Triplex RT-PCR detects rubella virus (p150 and E1 regions) with in-reaction quality control.•98.4 % sensitivity with 62 confirmed rubella positive samples.•100 % specificity with 165 rubella negative archival clinical samples.•Verified detection of nine genotypes including predominant 1E, 1G, 1J, 2B and vaccine (1a).•Lack of cross-reactivity demonstrated with eleven other viruses. Rubella surveillance in elimination setting relies on rapid molecular detection of the virus. In this study a multiplex real-time RT-PCR assay for the detection of rubella virus was validated. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the rubella viral coding regions for envelope glycoprotein E1 and non-structural p150 protein, and an endogenous control (human RNaseP). Using dilution series of synthetic RNAs, the limits of detection were determined to be at least 50 copies of rubella RNA. The assay is reproducible with low intra-assay and inter-assay coefficients of variation for both the E1 and the p150 targets. After testing 62 confirmed rubella positive and 165 rubella negative archival clinical samples, the sensitivity and specificity of the multiplex assay were 98.4 and 100 %, respectively. No cross reactivity was identified with clinical specimens positive for eleven other viruses. This multiplex assay successfully detected nine viral genotypes including the predominant genotypes 1E, 1G, 1J, and 2B as well as the 1a vaccine genotype.