18s rrna endogenous control primer probe set

All primers and probes for detection of viral RNA were designed and obtained from Integrated DNA Technologies (IDT) utilizing the PrimerQuest Tool. Primer/probe sets contained a double-quenched ZEN/IBFQ probe with a 6-FAM fluorescent dye attachment at the 5′ end. Capsid primer/probe set, initially described by Julander et al. [38 (link)] was used and is represented in Table 1. Quantitative data for nsP3 is represented for each non-structural RT-PCR figure and the primer/probe set is represented in Table 1. 18S rRNA endogenous control primer/probe set was obtained from ThermoFisher (4333760T). cDNA specific to negative-strand RNA for VEEV TC-83 was generated using a forward primer containing a T7 promoter sequence attached at the 5′ end and a high-capacity cDNA reverse transcription kit (4368814, ThermoFisher) per the manufacturer’s instructions. For qPCR of negative-strand viral RNA, forward primer specific to the T7 promoter sequence and reverse primer specific to VEEV TC-83 were utilized. All primer sets for negative-strand viral RNA quantitation are represented in Table 1.

All primers and probes were designed and obtained from Integrated DNA Technologies (IDT) using the PrimerQuest Tool. Primer/probe sets targeting the capsid and nsP3 region of the viral genome, which have previously been described [12 (link)]. Briefly, the sets contained a double-quenched ZEN/IBFQ probe with a 6-FAM fluorescent dye attachment at the 5′ end. A 18S rRNA endogenous control primer/probe set was obtained from ThermoFisher (4333760T). Amplification of the negative-strand TC-83 RNA has previously been described [12 (link)]. cDNA specific to negative-strand TC-83 RNA was generated using a T7 promoter sequence tagged at the 5′ end of the negative-strand and the high-capacity cDNA reverse transcription kit (ThermoFisher, 4368814), according to the the manufacturer’s instructions.