Supplementary Figure S5: Improved SARS-COV-2 PCR detection and genotyping with double-bubble primers
Intra-assay variation of RT-qPCR using D-B primer mix #8. cDNA equivalent to 103 copies of synthetic SARS-COV-2 RNA was amplified by standard real-time RT-qPCR using TqM buffer mix+ UDP (Applied BioSciences) with VIC-TqM probe #12 in 10 replicates. At the end of the reaction, samples were resolved on 1.8% agarose gel (insert in the real time amplification plot). Ct Mean± SD= 31.950± 0.140; n=10. Coefficient of variation= 0.437%. Agarose gel (1.8%) insert: Lane 1- 100bp DNA ladder. Lanes 2- 11- ten replicates of the PCR reaction. Expected amplicon size- 139bp. It should be noted that the agarose gel does not represent an accurate quantitative evaluation of the reproducibility since the samples were taken at the end of the PCR reaction after 40 cycles which are not at the exponential phase of the reaction and used here primarily for verification of the expected amplicon size -139bp. qPCR was performed using standard conditions. PCR was performed using standard conditions.