Nature 502, 100–104 (2013); doi: 10.1038/nature12519; corrigendum Nature 514, 654 (2014) Here we provide additional information about the specificity and validation of the simian immuno deficiency virus (SIV) DNA and RNA assays reported in this Letter. As described, we used quantitative PCR and quantitative PCR with reverse transcription (qPCR and qRT–PCR, respectively) assays targeting sequences in the SIV gag gene for quantification of SIV RNA and DNA. For measurements of plasma viraemia (SIV RNA), we used a standard qRT–PCR assay that has been extensively used in the field. For measurements of cell-and tissue-associated SIV DNA and RNA, we used nested qPCR and qRT–PCR assays. In our Letter, we report the evaluation of rhesus cytomegalovirus (RhCMV)-based vaccine vectors that include one vector with an SIV gag sequence insert. As shown in Fig. 1, sequence identity between the SIV gag insert in the RhCMV/Gag vector and the SIV gag-targeted primer and probe sequences for the standard qRT–PCR assay used for plasma SIV RNA measurements (sGAG21, sGAG22 and sGAG23 probes) raises the possibility that transcripts from the vaccine insert could have been detected by the assay we used.

For the nested assay, designed mismatching of the outer reverse primer (SIVnestR01) and the codon-optimized portion of the vaccine gag insert sequence and specific priming of the reverse transcription step of the qRT–PCR assay is intended to prevent potential cross reactivity. Biological features of the RhCMV vectors used are expected to minimize the prospects of detection of vaccine insert-derived SIV gag RNA in plasma or insert-derived SIV gag DNA or RNA sequences in cell or tissue samples.