Supplementary Figure S8: Improved SARS-COV-2 PCR detection and genotyping with double-bubble primers

Using D-B primers with non-hot-start taq-polymerases for RT-qPCR under fast conditions. Upper panels: Real-time qPCR using D-B primers #8, non-hot-start taq-polymerase (NEB), cDNA from reverse-transcribed synthetic SARS-COV-2 RNA “N” as template, VIC-TqM-probe #12, and added ROX for internal calibration. Samples assayed in duplicates under fast conditions and 35x cycles. (A) amplification plot. Ct values: cDNA duplicate- 27.6 and 26.7, NTC- undetermined. (B) 5% agarose gel of PCR products, lane 1- ULR ladder, lanes 2, 3- cDNA from reverse-transcribed synthetic SARS-COV-2 RNA “N”, lane 4- empty, lanes 5, 6- NTC. Lower panels: One-tube RT-qPCR using D-B primers #8, non-hot-start taq-polymerase (FroggaBio), iScript reverse-transcriptase (Bio-Rad) synthetic SARS-COV-2 RNA as template, VIC-TqM- probe #12, added 0.2 mM dNTPs, and added ROX dye for internal calibration. (C) amplification probe. Ct values: RNA- 25.12, NTC- undetermined. (D) 5% agarose gel of PCR products, lane 1- ULR ladder, lane 2- synthetic SARS-COV-2 RNA template, lane 3- NTC. RT- qPCR was performed under fast conditions.