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. 2020 Sep 3;295(46):15438–15453. doi: 10.1074/jbc.RA120.015434

Figure 3.

Figure 3.

Performance of GT RT-qPCR Master Mix. RT-qPCR was performed with Georgia Tech thermal cycling conditions (Table 2) GT-Master Mix components (Table 4), using ATCC synthetic viral RNA (ATCC® VR-3276SD™) template, and Ct values were determined using a threshold of 0.1, unless otherwise noted. A, effect of CHAPSO (0.1%) and BSA (0.5 mg/ml) on GT-Master Mix (GT-MM) performance with IDT N1 primers and 50,000 copies of synthetic viral RNA. The no-template control did not amplify. B, performance of GT multiplex primers and probes with 50,000 copies of viral RNA with GT-Master Mix, compared with TaqPath, and effect of trehalose (9.5%) added to GT-Master Mix. C, performance of GT-Master Mix with IDT N1 primers and 500 copies of synthetic viral RNA, compared with TaqPath, after three freeze/thaw cycles (6 days of storage) at 2× concentration. Inset, Ct for GT-Master Mix and TaqPath over 6 days of storage. D, qPCR efficiency (E = 10(−1/slope) − 1) using autothreshold. GT-Master Mix and GT multiplex primers (N1 and N2 FAM readout, blue): 91.6%; GT-Master Mix and GT singleplex primers (brown): 87.8% for GT-N1 primer/probe (diamond), 77.1% for GT-N2 primer/probe (triangle), 86.4% for GT-RP primer/probe (inverted triangle), and 100.3% for TaqPath with GT-N1 primer/probe (red). Singleplex RT-qPCRs were performed with a mix of full-length viral RNA and HEK293T total RNA.