Quantstudio 3d digital pcr master mix v2

The extracted DNA was diluted with RNAase-free water. The reaction mixture included 33 ng of DNA, RNAase-free water, 7.5 µL QuantStudio™ 3D Digital PCR Master Mix v2 (Applied Biosystems), and primers (made up to a volume of 14.5 µL) and was applied to the QuantStudio™ 3D Digital PCR 20 K Chip Kit v2 (Applied Biosystems). The primer sequences are as follows: forward primer, 5′-AAGCGCGTCTAGGTGGTTATGT-3′; reverse primer, 5′-TGTAGTTCCGCTTACCTCTCCAG-3′; and probe, 5′-FAM-CACGCAATACAGTTGAGCCCTGCATT-3′ (Applied Biosystems). The ProFlex PCR System (Applied Biosystems) thermocycler was used for amplification. DNA was amplified by initial denaturation at 96 °C for 10 min, followed by 39 cycles of 56 °C for 2 min, 98 °C for 30 s, 60 °C for 2 min, and a final hold at 10 °C. The chips were read using the QuantStudio™ 3D Digital PCR instrument (Applied Biosystems) and analysed using the QuantStudio 3D Analysis Suite Software (Applied Biosystems).

PCR experiments were performed according to the manufacturer’s instruction (Applied Biosystems, Foster City, CA, USA). For PCR experiments, 80 µL of the PCR mixture solution consisted of 45 µL of a commercial master mix (Quant Studio 3D Digital PCR Master Mix V2, Applied Biosystems, Foster City, CA, USA), 9 µL of forward primer (5′-TGGTGCTGGTTCTGATAAAGGAG−3′), 9 µL of reverse primer (5′-GAATCTGCATCAGAGACAAAGTCA−3′), 9 µL of FAM probe, and 8 µL of cDNA template (final concentration: 104 copies/µL). After photothermal bonding between PMMA-based plastic lab-on-a-chip, 20 µL of the PCR solution was pipetted into the PMMA-based plastic lab-on-a-chip and the inlet was sealed with a self-adhesive PCR sealing tape (4titude, Dorking, UK) to prevent water evaporation. The PMMA-based plastic lab-on-a-chip was then placed onto a thermocycler (DH200, BioD, Gwangmyeong, Korea) and PCR was conducted with 35 cycles of denaturation at 95 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. To analyze the fluorescence intensity, the PMMA-based plastic lab-on-a-chip was imaged using an inverted fluorescence microscope and the images were analyzed using Image J software.

Primers designed for RT-qPCR were used in these experiments. Taqman probes targeting C. auris FKS1 and FKS2 genes were designed using Beacon Designer 7.0 software (Premier Biosoft, Palo Alto, CA) and synthesized by Merck, Sigma-Aldrich (Madrid, Spain). Sequences of primers and probes used in this study are detailed in Table 2. The number of FKS1 and FKS2 cDNA molecules was quantified using QuantStudio^TM 3D Digital PCR System (Applied Biosystems, A29154). Conditions for digital PCR were as follows: 1× QuantStudio 3D Digital PCR Mastermix v2 (Applied Biosystems, 4482710), 0.4 µM forward and reverse primer (FKS1 or FKS2), 0.2 µM MB probe and 50 ng of cDNA (see above), in a final volume of 14.5 μl. Reactions were placed on a QuantStudioTM 3D Digital PCR 20 K Chip v2 (Applied Biosystems, A26317) automatically using QuantStudio^TM 3D Digital PCR Chip Loader (Applied Biosystems, CA, USA), and amplified following thermal cycling conditions recommended by manufacturer’s protocol. Applied Biosystems™ QuantStudio™ 3D AnalysisSuite™ Cloud Software was used for the analysis of data derived from the QuantStudio 3D Digital PCR instrument.

Viral RNA production was measured by RT–PCR and DRS analysis. An equal amount of total cellular RNA for each sample was used for RT and complementary DNA generation. The QuantStudio 3D Digital PCR System (Applied Biosystems) was used with appropriate consumables provided by the manufacturer, including the QuantStudio 3D Digital PCR Master Mix v.2 and TaqMan 5′-6 FAM or VIC probe (Applied Biosystems). The primers used are listed in Supplementary Table 9.

Digital PCR (dPCR) was performed using a QuantStudio 3D Digital PCR System (Applied Biosystems) in 14.5 μL reaction mixture containing 2 ng/μL genomic DNA template, 1× QuantStudio 3D Digital PCR Master Mix v2, 0.45 μM forward and reverse primers, 0.125 μM TaqMan probe, and 1× TaqMan copy number reference assay RNase P (Applied Biosystems). Sequences of probes and primers are shown in Table S2. The reporter dyes of the TaqMan probes hybridizing to provirus and RNase P were FAM and VIC, respectively. The cycling conditions were 96°C for 10 min, 39 cycles of 98°C for 30 s, and 60°C for 2 min, 60°C for 2 min, and infinite hold at 10°C. The PVL was calculated as: PVL = 100 × (provirus copy number × 2)/RNase P copy number.

Prior to implementation, dPCR was validated using the same standard curve samples as for qPCR validation. Quality threshold (Q_T) and fluorescence threshold (F_T) were determined based on the comparison of 14 negative template controls (i.e., negative matrix controls as determined by the plate count method and qPCR; NTC), 10 water no template controls (PCR grade water, WNTC), and 10 positive controls for increased stringency. The observed copy number/μl in dPCR was converted to CFU/ml taking into account all dilution factors.
Absolute quantification of C. jejuni with dPCR was performed in 15-μl reactions consisting of 7.5 μl QuantStudio 3D Digital PCR Master Mix v2 (Applied Biosystems by Thermo Fisher Scientific, USA), 3 μl DNA, 3.75 μl of PCR grade water, and ccoN primers and probes at previously described concentrations (Toplak et al., 2012 (link)). A 14.5 μl aliquot of each reaction mix was loaded onto dPCR chips from QuantStudio 3D Digital PCR 20K Chip Kit v2 using QuantStudio 3D Digital PCR Chip Loader. Amplification was performed in the PCR cycler ProFlex 2 × flat PCR System (Applied Biosystems by Thermo Fisher Scientific, USA) according to manufacturer's instructions. NTC and a positive control were included in each run. After amplification, all chips were analyzed using QuantStudio 3D AnalysisSuite 3.0.3.