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. Author manuscript; available in PMC: 2024 Oct 8.
Published in final edited form as: COVID. 2023 Jul 21;3(7):1031–1041. doi: 10.3390/covid3070075

Figure 2. Matrix Testing and Optimization of Mastermix and Sample Volume A.

Figure 2.

Optimization of the multiplex SARS-CoV-2 involved testing assay performance in water, PBS and saliva showed similar sensitivity in all three solutions at 100,000 copies of heat-inactivated SARS-CoV-2 virus (ATCC). Based on this data we proceeded with a PBS-based nasal swab test as it maintained similar sensitivity to sample in water and was easier and safer for us to collect, inactivate and test. B. After selecting a PBS-based assay we optimized the amount of sample added to the qRT-PCR reaction. The N1 primer/probe set performed well across all conditions but there were some sensitivity issues with the N2 primer/probe set with high sample input. We decided to stick with 3 μL sample input; this gave us the lowest average Ct values across primer/probe sets and would allow for plenty of residual sample for repeat testing, variant testing and sequencing. C. We also optimized the amount of Taqpath Master Mix added to the reaction and determined 3 μL of Taqpath produced optimal Ct values across primer/probe sets.