PrimeTime™ One-Step 4X Broad-Range Master Mix

Figure 1. PrimeTime™ One-Step 4X Broad-Range Master Mix demonstrates comparable amplification of viral analytes between extracted and non-extracted human saliva samples. RT-qPCR probe assays containing probes and primers specific for FluA, FluB, SARS-CoV-2, and RNase P were used to amplify 30 copies of AccuPlex™ SARS-CoV-2, Flu A/B and RSV Verification Panel (SARS-CoV-2 data shown) material that had been extracted (dark blue) or direct amplified (light blue). The AccuPlex™ analyte verification panel was first diluted into negative human saliva matrix before RNA extraction or direct specimen amplification, and RNA extractions were performed automated using the KingFisher™ Flex 96 and MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit. Reactions were amplified and analyzed using the QuantStudio 7 Flex instrument according to the manufacturer's instructions. Experiments were performed with multiple replicates (n = 20) for each condition.

Figure 2. PrimeTime One-Step 4X Broad-Range Master Mix demonstrates comparable amplification of viral analytes between extracted and non-extracted human nasopharyngeal samples. RT-qPCR probe assays containing probes and primers specific for FluA, FluB, SARS-CoV-2, and RNase P were used to amplify 30 copies of AccuPlex™ SARS-CoV-2, Flu A/B and RSV Verification Panel (SARS-CoV-2 data shown) material that had been extracted (dark blue) or direct amplified (light blue). The AccuPlex™ analyte verification panel was first diluted into negative human nasopharyngeal (NP) matrix before RNA extraction or direct specimen amplification, and RNA extractions were performed automated using the KingFisher Flex 96 and MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit. Reactions were amplified and identified using the QuantStudio 7 Flex instrument according to the manufacturer's instructions. Experiments were performed with multiple replicates (n = 20) for each condition.

Figure 5. PrimeTime One-Step 4X Broad-Range Master Mix has a higher tolerance to heparin inhibition than three other commercially available master mixes. RT-qPCR probe assays containing probes and primers specific for Flu A (shown), Flu B, SARS-CoV-2, and RNase P genes were used to amplify RNA template in the presence of heparin concentrations ranging from 0.78−25 U/mL. Each master mix was used according to the manufacturer’s instructions. RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 6, per inhibitor concentration per master mix). Error bars represent standard deviation. *Indicates reactions where no amplification occurred in the presence of heparin.

Figure 6. PrimeTime One-Step 4X Broad-Range Master Mix has a higher tolerance to hematin inhibition than three other commercially available master mixes. RT-qPCR probe assays containing probes and primers specific for Flu A (shown), Flu B, SARS-CoV-2, and RNase P genes were used to amplify RNA template in the presence of hematin concentrations ranging from 1.88−60 µM. Each master mix was used according to the manufacturer’s instructions. RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 6, per inhibitor concentration per master mix). Error bars represent standard deviation. 

Figure 7. PrimeTime One-Step 4X Broad-Range Master Mix has a higher tolerance to humic acid inhibition than two out of three other commercially available master mixes. RT-qPCR probe assays containing probes and primers specific for Flu A (shown), Flu B, SARS-CoV-2, and RNase P genes were used to amplify RNA template in the presence of humic acid concentrations ranging from 1.88−60 ng/µL. Each master mix was used according to the manufacturer’s instructions. RT-qPCR reactions were run on the QuantStudio 7 Flex (n = 6, per inhibitor concentration per master mix). Error bars represent standard deviation.