Strand-specific qPCR Standard Curves for ONNV and CHIKV

To generate strand-specific standard curves for ssqPCR, (+) and (−) strand RNA was transcribed in vitro from a plasmid containing a portion of the nsP1 gene from either ONNV or CHIKV. The plasmids pblue-nsP1 (ONNV) and pblue-nsP1 (CHIKV) were produced by cloning the 5′ terminal 853 and 669 nucleotides of the respective viral nsP1 gene into pBluescript II SK (−) (Stratagene). Minus strand RNA was synthesized with T7 RNA polymerase from HindIII-digested plasmid templates in a standard in vitro transcription reaction. Positive strand RNA was synthesized with SP6 RNA polymerase from KpnI-digested plasmid templates in a standard in vitro transcription reaction. The RNA generated during the in vitro transcription reactions was isolated with TRI Reagent RT®, as per manufacturer's instructions. The absence of template DNA was confirmed through PCR. The concentration of RNA transcripts was determined with a NanoDrop spectrophotometer (Thermo Scientific). The cloned ONNV nsP1 gene fragment has a molecular weight of 273,545 g/mol, while the cloned CHIKV nsP1 gene fragment has a molecular weight of 214,574 g/mol. One µg of RNA transcribed from pblue-nsP1 (ONNV) equals approximately 2.2×10¹² molecules, while one µg of RNA transcribed from pblue-nsP1 (CHIKV) equals approximately 2.8×10¹² molecules.