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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: J Virol Methods. 2019 Jul 14;272:113701. doi: 10.1016/j.jviromet.2019.113701

Figure 4. Schematic diagram of modified RT-qPCR with tagged RT primers and modified PCR primer sets.

Figure 4.

Left panel shows schematic diagram of RVFV RNAs and binding sites of tagged RT primers to viral RNA segments. cDNA synthesis is performed using a tagged RT primer complementary to either genomic or antigenomic viral RNA segments. The tagged non-viral sequence, shown in red, is attached to the 5’-end of each strand-specific RT primer. For genomic segments, the tagged genomic RT primers bind to the 3’-end of these RNAs. Note that the tagged genomic RT primer binds to the 3’-end of genomic S segment, but not N mRNA, as the RT primer is in the same orientation as N mRNA. The same principle is applied for the tagged RT primer for antigenomic S segment, which does not bind to NSs mRNA. The tagged RT primers for antigenomic M and L segments bind downstream of the transcription termination sites of these segments. The dashed lines indicate the 3’-ends of L and M mRNAs compared to the full-length antigenomic segments. The right panel outlines the PCR of the cDNA products by using the forward PCR primer (red arrows), which is the tag portion of the RT primer, and a strand-specific reverse PCR primer (black arrows). Use of the tagged portion as a forward PCR primer ensures that only primer-driven tagged cDNAs undergo amplification.