Conventional RT-PCR methods are performed to amplify the 739-nt region of the E1 gene for assignment of rubella genotypes. Many variations of RT-PCR have been used to detect rubella RNA in clinical specimens for case confirmation. Currently, real-time RT-PCR is the most commonly used confirmatory method for rubella in the GMRLN laboratories. Several laboratories in GMRLN have developed real-time RT-PCR assays that target different regions and amplify amplicons of various lengths of the rubella genome [1,11-14]. A different system, the RT-loop-mediated isothermal amplification (RT-LAMP) has been described for rubella detection [15].

The original protocol for rubella real-time RT-PCR, included in training workshops provided by CDC (Atlanta), amplified a 185-nt region that lies within the 739-nt region of the E1 gene [11,16]. A modification of the protocol has been provided to improve the sensitivity of the assay for the rubella viruses in genotype 2B. The modification consists of the addition of second reverse primer, based on the consensus sequence of 151 genotype 2B rubella viruses. The additional reverse primer was described in a study of rubella viruses in Uganda in 2014 [17]. The kits produced at the CDC that amplify the 185-nucleotide region within the E1 gene include two synthetic positive controls, a high copy control and a low copy control. Primers and probe for the reference gene, RNase P, are also included. The protocol for the conventional RT-PCR for rubella detection using the 3-primer system is provided in Annex 6.4.

While the CDC rubella real-time RT-PCR assay described above may still be in use, an alternative target has been identified for a real-time assay that further improves detection of 2B genotype viruses. This assay can detect as few as three to seven copies of rubella-specific RNA of both clade 1 and clade 2 viruses. The assay targets the 5’ terminus of the rubella genome and amplifies a 154- nucleotide region of the p150 gene. This assay was used in an investigation of a rubella outbreak in Romania [18]. The protocol for the CDC rubella real-time RT-PCR is available in pdf format (Annex 6.5). Additional assays that have been described include a one-step TaqMan assay that targets a highly conserved region in the other rubella non-structural gene (p90 gene) [14].