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. 2014 Dec 5;213:26–37. doi: 10.1016/j.jviromet.2014.11.015

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Copyright © 2014 Elsevier B.V. All rights reserved.

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Fig. 1 — The relative frequency of mRNA transcription, genome organization for each vector, the scheme for the mRNA specific RT-qPCR, and the binding sites for qPCR primers and probe for the SIV gag gRNA and mRNA assays are depicted. (A). Transcriptional attenuation resulting in a gradient of mRNA synthesis such that the gag message is more abundant than the N message in the rCDV-SIVgag1 vector; the rCDV vector is a Vero cell adapted, Onderstepoort live-attenuated CDV vaccine (Galaxy D, Schering-Plough Animal Health Corporation, Omaha, NE, USA) obtained through a commercial source; and the rCDV-SIVgag1 vector with the Gag gene in the first position of the genome and a unique UAUAAU sequence common to CDV N and SIV gag genomes. (B). Diagram of the RT step used in the singleplex or duplex assay to synthesize cDNA from mRNA. The CDV-specific mRNA RT-primer that binds to a unique UAUUA sequence common only to CDV N and SIV gag mRNA transcripts, has a 22-base adapter sequence at the 5′ end followed by a 20-base oligo-dT sequence, and a 3′-terminal 5-base anchor (ATAAT). (C) Tagged cDNA generated with this primer is then PCR amplified with CDV N and SIV-gag forward primers and labeled probes and the CDV sequence tag-reverse primer that corresponds to the adapter sequence, as described in Section 2. (E). The black box from 256–339 Nt indicates the binding site for the qPCR primers () and probe () for the Gag specific gRNA RT-qPCR assay and the 1500–1669 Nt indicates the amplicon and the binding site for qPCR forward primer () and probe () used in the Gag specific mRNA RT-qPCR assay.

Inline graphic — The relative frequency of mRNA transcription, genome organization for each vector, the scheme for the mRNA specific RT-qPCR, and the binding sites for qPCR primers and probe for the SIV gag gRNA and mRNA assays are depicted. (A). Transcriptional attenuation resulting in a gradient of mRNA synthesis such that the gag message is more abundant than the N message in the rCDV-SIVgag1 vector; the rCDV vector is a Vero cell adapted, Onderstepoort live-attenuated CDV vaccine (Galaxy D, Schering-Plough Animal Health Corporation, Omaha, NE, USA) obtained through a commercial source; and the rCDV-SIVgag1 vector with the Gag gene in the first position of the genome and a unique UAUAAU sequence common to CDV N and SIV gag genomes. (B). Diagram of the RT step used in the singleplex or duplex assay to synthesize cDNA from mRNA. The CDV-specific mRNA RT-primer that binds to a unique UAUUA sequence common only to CDV N and SIV gag mRNA transcripts, has a 22-base adapter sequence at the 5′ end followed by a 20-base oligo-dT sequence, and a 3′-terminal 5-base anchor (ATAAT). (C) Tagged cDNA generated with this primer is then PCR amplified with CDV N and SIV-gag forward primers and labeled probes and the CDV sequence tag-reverse primer that corresponds to the adapter sequence, as described in Section 2. (E). The black box from 256–339 Nt indicates the binding site for the qPCR primers () and probe () for the Gag specific gRNA RT-qPCR assay and the 1500–1669 Nt indicates the amplicon and the binding site for qPCR forward primer () and probe () used in the Gag specific mRNA RT-qPCR assay.