Ddpcr multiplex supermix

Manufactured by Bio-Rad

Sourced in United States

The DdPCR Multiplex Supermix is a ready-to-use reaction mix designed for digital droplet PCR (ddPCR) applications. It contains all the necessary components, including a thermostable DNA polymerase, dNTPs, and buffers, to amplify and detect multiple target sequences simultaneously in a single reaction.

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DdPCR Supermix (116 citations)

8 protocols using ddpcr multiplex supermix

Quantitative ddPCR Detection of Viral Particles

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ddPCR was performed using a previously described duplex assay^{25 (link)} with the primers and probes given in Supplementary Table S3. VLPs were lysed by heating to 95°C for 5 min in a thermocycler before being added to ddPCRs. ddPCRs (20 μL) used ddPCR™ Multiplex Supermix (Bio Rad) with final primer and probe concentrations of 900 and 250 nM and a sample volume of 2 μL. Droplets were then generated using a QX200™ Droplet Generator (Bio Rad) and transferred to a PCR plate and sealed, all according to the manufacturer's instructions.
The reaction mixture was then thermocycled (95°C for 10:00, 40 cycles of 94°C for 0:30, and 60°C for 1:00, and a final enzyme inactivation step of 98°C for 10:00) on a BioRad C1000 touch PCR machine. Finally, droplets were read using the QX200 Droplet Reader (Bio Rad). Data were then analyzed using a Python implementation (https://github.com/mcrone/plotlydefinerain) of an online tool called Defining The Rain (https://definetherain.org.uk/) with added support for two color channels.

Crone M.A, & Freemont P.S. (2022). Simple Low-Cost Production of DNA MS2 Virus-Like Particles As Molecular Diagnostic Controls. Gen Biotechnology, 1(6), 496-503.

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Droplet Digital PCR for Microbial Quantification

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For most samples, ddPCR was performed using 1 μL of 1:100 diluted DNA isolated from solubilized sputum (Bio-Rad QX200 ddPCR system) using the ddPCR Multiplex Supermix (Bio-Rad) and previously validated primer and probe sets (16 (link)) adapted for ddPCR. Results were analyzed with QX software to determine target DNA concentrations. For low-abundance samples, up to 13 μL of undiluted DNA was used. Positive control (DNA isolated from bacterial cultures) and negative controls (no sample added) were run with each assay. Results from 16S rRNA gene ddPCR were divided by 4 to correct for 16S rRNA gene copy number. Extraction blanks had a median background level of 1.43 × 10³ (range 0 to 3.75 × 10³) 16S rRNA gene copies/mL and sputum had a median concentration of 1.3 × 10⁸ gene copies/mL.
ddPCR assays were validated by comparing DNA concentration measurements obtained from serial dilutions of each measured species using Qubit (Thermo Fisher Scientific) and ddPCR (see Supplemental Figure 5). The ddPCR threshold over which pathogens were considered present was set at greater than 200 genomes/mL, as no negative control (i.e., water or extraction blank sample) contained greater than 200 pathogen copies/mL. To calculate log reduction in genome copies/mL, negative samples were set to half the detection threshold (100 copies/mL).

Nichols D.P., Morgan S.J., Skalland M., Vo A.T., Van Dalfsen J.M., Singh S.B., Ni W., Hoffman L.R., McGeer K., Heltshe S.L., Clancy J.P., Rowe S.M., Jorth P, & Singh P.K. (2023). Pharmacologic improvement of CFTR function rapidly decreases sputum pathogen density, but lung infections generally persist. The Journal of Clinical Investigation, 133(10), e167957.

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Intestinal Microbiome Characterization for Crohn's Disease Biomarkers

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To identify potential non-invasive biomarkers from the characterization of the intestinal microbiome in various stages of CD, LEfSe (Linear Discriminant Analysis Effect Size) was used^{22 (link)}. In addition, the absolute quantification of Faecalibacterium prausnitzii, Escherichia coli, and the total bacteria species present was studied using the QX200 Droplet Digital PCR system (ddPCR; Bio-Rad Laboratories), following the ddPCR Multiplex Supermix (BIORAD) kit protocol^{5 (link),23 (link)–26 (link)}. For this, the appropriate primers and probes were selected to perform a triplex PCR together with the optimization of their concentrations and thermal programs^{5 (link),23 (link)–26 (link)}. The QuantaSoft program version 1.7.4 was used to export the recorded amplitude data. The Faecalibacterium prausnitzii/Escherichia coli (F/E) ratio analysis was normalized using the following equation ([log10 (copies/µL F. prausnitzii) – log10 (copies/µL E. coli)] / [log10 (copies/µL ARNr 16S])^{27 (link)}_.

Sanchis-Artero L., Martínez-Blanch J.F., Manresa-Vera S., Cortés-Castell E., Valls-Gandia M., Iborra M., Paredes-Arquiola J.M., Boscá-Watts M., Huguet J.M., Gil-Borrás R., Rodríguez-Morales J, & Cortés-Rizo X. (2021). Evaluation of changes in intestinal microbiota in Crohn’s disease patients after anti-TNF alpha treatment. Scientific Reports, 11, 10016.

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Optimizing Thermal Gradient for Viral Quantification

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We ran a thermal gradient to evaluate the optimal annealing temperature. The temperature gradient (number of technical replicates for each temperature n = 3) ranged from 64 °C to 54 °C using the same DNA and primer/probe concentrations as for the single- and multiplex tests (10 pg µL⁻¹). We further tested different concentrations of DNA templates for giant virus and virophage (10 pg µL⁻¹, 1 pg µL⁻¹ and 0.1 pg µL⁻¹; number of technical replicates for each template n = 6) to determine the best range for quantification accuracy. DNA concentrations were selected based on previous preliminary experiments. We used the primer concentrations recommended by BioRad© for the ddPCR Multiplex Supermix (Table 2).

del Arco A., Fischer M, & Becks L. (2022). Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR. Viruses, 14(5), 1056.

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Digital Droplet PCR Quantification Protocol

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PCR mastermix was prepared by adding ddPCR Multiplex Supermix (Bio-Rad Laboratories), primers (950 nM), probes (250 nM), and DNA (350 ng) at a final volume of 20 µl. Next, QX200 ddPCR droplet generator (Bio-Rad Laboratories) was utilized to separate the DNA into 20,000 droplets, which were transferred to a 96-well plate and sealed to avoid evaporation using the PX1 PCR Plate Sealer (Bio-Rad Laboratories). Finally, the PCR was run on the C1000 Touch Thermal Cycler (Bio-Rad Laboratories) with the following thermal parameters: 10 min at 95 °C, 40 cycles comprising 30 s at 95 °C, 1 min at 61 °C, and 2 min at 72 °C, followed by enzyme inactivation at 98 °C during 10 min. Finally, PCR products were examined using the QX2000 droplet reader (Bio-Rad Laboratories) and analyzed with the QuantaSoft 1.6.6 software (Bio-Rad Laboratories).

Lamsfus-Calle A., Daniel-Moreno A., Antony J.S., Epting T., Heumos L., Baskaran P., Admard J., Casadei N., Latifi N., Siegmund D.M., Kormann M.S., Handgretinger R, & Mezger M. (2020). Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34+ HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin. Scientific Reports, 10, 10133.

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Absolute Quantification of ESR1, ESR2 and ACTB3

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To quantify absolute concentrations of ESR1, ESR2 and ACTB3 in each sample, a reaction mixture of 20 μl was prepared containing 5 ng cDNA, forward and reverse primers (100 nM each) and ddPCR Multiplex Supermix (Bio-Rad, catalogue number: 12005909). Droplets were generated using the QX200 Droplet Generator (Bio-Rad) and transferred into a 96-well plate for amplification. The plate was loaded into the QX200 Droplet reader (Bio-Rad) which analysed each droplet per sample for the presence or absence of the target DNA. The QX200 Quantasoft software was used to quantify the levels of ESR1, ESR2 and ACTB3 in each sample tested.

Augustine T.N., Buthelezi S., Pather K., Xulu K.R, & Stoychev S. (2024). Secretomics reveals hormone-therapy of breast cancer may induce survival by facilitating hypercoagulation and immunomodulation in vitro. Scientific Reports, 14, 1486.

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Screening Pentaplex Assay for EGFR Mutations

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The screening pentaplex is based on the previously described EGFR triplex assay, targeting Ex19Del, L858R, and L861Q. 14 For G719S, 1.8 mmol/L primers, 0.25 mmol/L G719S FAM-labeled mutant (MT) probe, and 0.25 mmol/L HEX-labeled wild-type (WT) probe (Bio-Rad Laboratories, Hercules, CA) were added. For S768I, 1.8 mmol/L primers and 0.5 mmol/L FAM-labeled MT probe (Bio-Rad Laboratories) were added. Per reaction, 5 mL ddPCR Multiplex Supermix (Bio-Rad Laboratories) was used.

de Kock R., van den Borne B., Youssef-El Soud M., Belderbos H., Brunsveld L., Scharnhorst V, & Deiman B. (2021). Therapy Monitoring of EGFR-Positive Non-Small-Cell Lung Cancer Patients Using ddPCR Multiplex Assays. The Journal of molecular diagnostics : JMD, 23(4).

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Droplet Digital PCR for CMV Quantification

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The ddPCR reaction was performed using ddPCR Multiplex Supermix (Bio-Rad, Hercules, CA, USA), using primers and probes as described in Table 1, with 10 units of Hind III and 10 µL of template. The final concentration of the primers was 900 nM and 250 nM for the UL55-Hex [8 (link)] and UL83-FAM reactions, respectively. After droplet generation with the QX200 Droplet Generator, the ddPCR was run using a C1000 Touch thermal cycler with the cycling conditions as follows: 95 °C for 10 min, 45 cycles of 94 °C for 30 s, 60 °C for 1 min, and 98 °C for 10 min. The results were read in the QX100 Droplet Reader and analyzed with the QuantaSoft Analysis Pro software (v1.0, Bio-Rad, Hercules, CA, USA).

Hernandez-Alvarado N., Bierle C.J, & Schleiss M.R. (2023). Droplet Digital PCR (ddPCR) Does Not Enhance the Sensitivity of Detection of Cytomegalovirus (CMV) DNA in Newborn Dried Blood Spots Evaluated in the Context of Newborn Congenital CMV (cCMV) Screening. International Journal of Neonatal Screening, 10(1), 1.

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