Quantstudio absolute q digital pcr system

The Omicron RT-dPCR assays (Omn143) was performed using the QuantStudio Absolute Q Digital PCR System (Applied Biosystems, Waltham, MA, USA) in 10 μL reaction mixtures (including 10% overage) containing 2.5 μL of 4× Combinati one-step RT-dPCR MasterMix, 0.4 μL of each 10 μM primer (Omn143-Fwd and Omn143-Rev; Table S2), 0.2 μL of 10 μM probe (Omn143 Probe; Table S2), and 6.5 μL of sample RNA. Reaction mixtures were loaded into QuantStudio Absolute Q MAP16 plates and overlaid with 15 μL of isolation buffer. The assay was performed with thermal cycling conditions: reverse transcription at 50°C for 10 min, activation/denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 5 s and annealing/extension at 54°C for 15 s. Assay validation was performed in three independent experiments using synthesized gBlock gene fragments (Integrated DNA Technologies, Coralville, IA, USA): Omicron BA.1 control and Delta control (Table S2). Microreaction chambers that passed QC, as determined by ROX signal, were analyzed. FAM fluorescence intensity threshold to call Omicron positive detection per microchamber was set at 10,000 (more than two standard deviations above water and Delta negative controls). Absolute quantification (copies) was reported.

Trueprep eluate that tested positive for MTB on the Molbio MTB Plus assay was quantified by dPCR. Protein antigen b (PAB) was targeted for quantification. PAB primers and probes were designed in-house (Table S2). The final reaction mix included 1.8 µL of Absolute Q DNA Digital PCR Master Mix (ThermoFisher, #A52490), 0.45 µL of 20× assay, 2.75 µL of nuclease-free water, and 6 µL of Trueprep eluate to bring the final reaction volume to 9 µL. dPCR was run on the QuantStudio Absolute Q Digital PCR System (ThermoFisher). A standard threshold for each plate was set equal to the automatically determined threshold for the positive control.