Guide to multiplexing dPCR assays

What is multiplex digital PCR?

Multiplexing in PCR enables the detection of more than one target molecule or sequence in a single reaction. Multiplexing with digital PCR (dPCR) can be achieved by varying the type of fluorophores used. In traditional multicolor multiplexing, targets are differentiated using one probe per target where the probe is conjugated with dyes of different emission spectra. Most dPCR instruments offer the detection of different fluorophores on at least two dedicated detection channels. Some dPCR systems enable 5-color multiplexing for clear discrimination of more targets in the same reaction.

Advantages of dPCR multiplexing

Multiplexing in dPCR has been a growing topic of interest, due to the many benefits of multiplexing with a dPCR system. The approach can be used to:

Analyze multiple targets in a single reaction
Reduce technical errors, such as pipetting inaccuracies
Increase chances of target detection
Reduce the amount of sample needed for analysis
Provide direct internal control of an individual reaction
Save time and reagents to reduce overall cost

Multiplexing with digital PCR enables several applications that are not possible or easy to perform using single-colour assays. Some of these analyses include:

Copy number variants: With a duplex approach, pairing of target and reference genes to determine their ratio
Biallelic variation of single nucleotide variants or small insertions/deletions: Duplex approach with two hydrolysis probes, visualized using two colour plots (1)
Genotyping: Counting partition numbers from single and double positive clusters (1)

To obtain the full benefits of digital PCR multiplexing, consider a digital PCR system with advanced multiplexing capabilities.

A digital PCR instrument with high multiplexing power makes it possible for you to analyze multiple targets in one sample with high sensitivity and reproducibility. For example, the QIAcuity Digital PCR System exists in several configurations to fit your need for sample and target analysis.

The nanoplate digital PCR advantage for multiplex reactions

Multiplex digital PCR is a preferred approach, because the method is more flexible, sensitive and specific than multiplex qPCR. Multiplexing by qPCR might also require more complicated methods for calculating absolute values and the method is more susceptible to interference of multiple PCRs in one reaction.

Nanoplate digital PCR offers distinct advantages over other types of digital PCR, including short run times, high precision and sensitivity, and multiplexing capabilities. Because nanoplates rely on individual PCR reactions, rather than analysis of bulk droplets, it is possible to find rare targets (<10-20 copies/reaction).

Nanoplate digital PCR can accommodate a variety of sample matrices, enabling tittering at more stages of AAV production. The QIAcuity Digital PCR System also enables independent users to run multiple assays in parallel for a boost in productivity (29).

High-throughput and multiplexing features of the QIAcuity Digital PCR System

	QIAcuity One	QIAcuity Four	QIAcuity Eight
Detection channels (multiplexing)	2 or 5	5	5
Number of samples analyzed in one workday	Up to 384	Up to 672	Up to 1248
Recommended dyes	FAM; VIC, HEX; TAMRA; ROX; Cy5

What does 5-plex dPCR data look like?

In the figure on the left, 10 ng (A, top and bottom panels) and 1 ng (B, top and bottom panels) of cDNA was used as template input amount for the dPCR reaction. Five assay targets (ERBB2, EGFR, CDKN2A, KDR and CDK1) were measured in a 5plex reaction in a QIAcuity Nanoplate 8.5K 96-well using the QIAcuity One, 5plex System. The probes were labeled with FAM, HEX, TAMRA, ROX and Cy5, respectively. The 2D scatter plots show a clear separation of the single assays in a multiplex setup.

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Applications of multiplex dPCR

Multiplex dPCR assays can support a variety of digital PCR applications. These range from cell and gene therapy, to food fraud investigations, to AAV characterization, to microbial detection, to an assay for copy number variation.

Multiplex dPCR assays for food applications

Multiplex digital PCR assays have numerous uses in the food industry. In food regulatory control and quality assurance, multiplex dPCR assays can be applied to identify animal species and to detect the origin of meat and meat products, fish species and other species (2, 3). For example, with digital PCR multiplexing, you can successfully distinguish the origin of pig, camel, sheep, donkey, goat, cow and chicken in a single reaction (2).

Multiplexing by dPCR could also be used for GMO testing and quantification of transgenes using transgene-endogene multiplex PCR reactions (4, 27). In one study, duplex dPCR assays were used to compare levels of the MON810 transgene with those of an HMG reference gene in maize feed samples. The multiplex dPCR assay achieved a sensitivity of five target DNA copies with better repeatability and tolerance to seed powder inhibitors than qPCR (5).

Food fraud is another major application of multiplexing with digital PCR, for example, in the detection of animal-derived ingredients in processed vegetarian or vegan products. A multiplex dPCR assay based on the amplification of the mitochondria cytochrome b gene, specific to most animals, and chloroplast DNA, specific to plant species, can be used to detect animal-derived ingredients in vegetarian foods (6).

Lastly, dPCR can be used to detect serious foodborne diseases. With multiplexing by digital PCR, you can simultaneously detect microbial pathogens, such as E. coli, L. monoytogenes, S. aureus and S. enetrica (7).

mericon GMO Detection and Quant GMO kits, mericon Ingredient Authentication kits, DNeasy mericon Food kitApplied testing, food, safety testing, collecting samples from corn

Multiplex dPCR in a copy number variation assay

In a copy number variation assay, multiplexing by dPCR can be used to pair target and reference genes to determine their ratio using a duplex approach.

In one study, digital PCR multiplexing was used successfully able to simultaneously detect gene mutations, gene fusions and gene duplications (8). Simultaneous copy number assessment of two major oncogenes in neuroblastoma with two normal diploid reference genes was also achieved, saving precious sample. The multiplexed dPCR assays displayed 100% specificity and sensitivity in the simultaneous detection of gene mutations, fusion and duplication (9).

Another example of a copy number variation assay with dPCR multiplexing involves genotyping of spinal muscular atrophy (SMA) samples. The study authors used multiplexing with digital PCR to quantify SMN1 and SMN2 copy numbers with RPPH1 as an internal reference gene control (10). Studies have also shown digital PCR multiplexing can be used to precisely and cost-effectively detect large deletions and duplications in the BRCA1 gene (11), as well as MET and HER2 amplification in non-small cell lung cancer (NSCLC) (28).

Another study developed and optimized multiplex dPCR assays for quantification of copy number alterations (CNAs) in 15 genomic DNA biomarkers from frozen normal and squamous cell carcinoma (SCC) samples (12).

3D Illustration of Chromosomal Translocation, Fusion Gene Explanation, 05/2017, (Illustration, 3D Illustration, Life science)

Curious to learn more on CNV analysis with dPCR?

Do so with our webinar "Multiplex analysis of Copy Number Variation in liquid biopsy using dPCR: preliminary data from patients with solid tumors".

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Quantification of mutations using multiplex dPCR assays

Multiplexing DNA targets by dPCR is beneficial for the quantification of somatic mutations, absolute allele quantification and rare mutation detection. Studies show that multiplex dPCR panels can be used for quantitative analysis of oncogene mutations in cell-free DNA (cfDNA) (13) and plasma (14, 15).

Evidence suggests it is worthwhile to find optimal primer and probe concentrations and optimal elongation temperatures in singleplex reactions. You should also check for primer set compatibility and the presence of rain in duplex reactions. You can then move on to combinations in more complex multiplex dPCR reactions.

qBiomarker Somatic Mutation PCR Arrays, 337021, tablet, mutation, DNA, iPad, GeneGlobe, (PCR, Computer/Laptop/Tablet, Hand/handling, Photography, Gene Expression and Function (GEF), Life science, GeneGlobe

Multiplexing with dPCR for microbial detection

Multiplexing with digital PCR is frequently used to detect microbial species. For example, multiplex dPCR assays can quickly test for bacterial pathogens, fungal pathogens, viral pathogens and related resistance genes (16, 17, 18, 19, 20). Reported detection sensitivities are as low as 50 copy units per mL with one study reporting the sensitivity of dPCR as 104 times higher than qPCR in detecting bacterial DNA in blood (21).

Another publication developed duplex dPCR and duplex qPCR assays to assess cyanobacterial species in natural environments. The authors found that qPCR was quick and economical, but dPCR offered higher accuracy, precision and resistance to PCR inhibition (22).

Inhibition and competition between targets are major challenges for multiplex qPCR, especially for environmental samples, where targets are present in low quantities with a large amount of non-target background DNA. Digital PCR multiplexing has been shown to accurately quantify at least two targets with a 1000-fold difference in concentration, providing a viable solution for these qPCR limitations (22).

Dirt, Laboratory, Agriculture, Medical Sample, DNA, Microbiology, Scientist, Biochemistry, microbial DNA, bacteria and fungi, DNeasy, PowerSoil Pro kit, close up of scientist looking at soil sample in a petridish

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Then check out our scientific poster on accurate and sensitive detection of microbial DNA and RNA targets using nanoplate dPCR.

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Multiplex dPCR assays in biopharma

In the biopharma industry, dPCR multiplexing supports the manufacturing and quality control processes for monoclonal antibodies (MABs), vaccines, cell and gene therapy and biosimilars (23, 24, 25, 26). Multiplex dPCR assays can contribute to multiple aspects of quality control, from contaminant detection to potency quantification. The method makes efficient quality control possible, as multiple contaminants can be tested simultaneously.

Multiplexing by dPCR is a good fit for applications requiring accurate relative quantification of a target compared to a housekeeping gene, or if the vector being developed delivers multiple therapeutic genes and either a silencing gene or gene editing function. Researchers working with material from patients can also obtain more information from a single sample, eliminating the need for resampling and reducing human error and reagent cost per reaction.

Considerations for accurate quantification with multiplex digital PCR

Optimize individual assays: Validate individual reactions prior to dPCR multiplexing (for example, check for occurrence of dimers)
Assess primers and probes: Check for potential interaction between your primers and probes
Carefully select dyes: Choose dyes for each assay carefully; select dyes with a bright fluorophore for low copy targets
Check for linked targets: Investigate potential for linked targets or higher-order target correlations; for example, use linkage-based assays to measure cis versus trans configuration of targets and potential structural rearrangements
Avoid cross hybridisation: If possible, design probe-to-target variations involving two or more nucleotide deletions or insertions to avoid probe mismatches
Minimize optical bleedthrough: Occurs if the fluorescence from a dye is picked up by more than one channel; if possible, try to make sure your conjugated dyes match the optical detection systems or adjust signal processing parameters in the analysis software
Reduce presence of rain: Rain describes the subset of partitions that are higher than the negative but lower than the positive partitions. You can reduce rain by re-assessing your template type, confirmation, integrity, assay specificity and presence of inhibitors in the reaction; consider setting the threshold position differently to avoid influence from rain on quantification

More tips for dPCR assay optimization

Gain more insights on designing, troubleshooting and optimizing dPCR experiments by exploring our dPCR assay development page.

Learn more

Getting started with multiplex dPCR

If you are interested in performing multiplex digital PCR, consider investing in a dPCR instrument with advanced multiplexing capabilities. There are also numerous dPCR kits and dPCR assays developed for multiplex digital PCR assays that could bring multiple benefits to both beginners and experts of multiplexing.

Your digital PCR instrument – There are many types of digital PCR systems available, but some offer more multiplexing options than others. The QIAcuity dPCR system, for example, offers the possibility to multiplex in up to 5 channels (plus one reference channel) providing the simplest way to multiplex and save time and reagents:

Channel	Excitation (nm)	Emission (nm)	Example fluorophores
Green	463 – 503	518 – 548	FAM
Yellow	514 – 535	550 – 564	HEX, VIC
Orange	543 – 565	580 – 606	TAMRA, ATTO 550
Red	570 – 596	611 – 653	ROX, Texas Red
Crimson	590 – 640	654 – 692	Cy5

Your multipurpose dPCR kits – Consider using dPCR kits developed for multiplex reactions. The QIAcuity Probe PCR Kits include special master mixes to quantify up to five targets with widely differing abundance on a QIAcuity Nanoplate. dPCR multiplexing saves you time, costs and sample material without affecting data quality or validity.
dPCR kits for contamination-free analysis – Specialized kits exist for applications that require ultra-clean master mixes that minimize contaminating background DNA. The QIAcuity UCP Probe PCR Kit is ideal for microbial analysis or for quality control applications, such as residual DNA testing. The kits offer high specificity and accuracy in quantifying gDNA or cDNA in singleplex or 5-plex dPCR assays.
Application-specific dPCR multiplex assays – To increase throughput and reduce costs according to specific applications, dPCR assays with different dyes (FAM, HEX, ROX, Atto 500 and Cy5) have been developed. These multiplex dPCR assays enable flexible experimental design and multiplex analysis of up to five targets in one reaction. Specific dPCR assays are available for CNV analysis, microbial detection, cell and gene therapy and LNA mutation assays for cancer-related mutations.

Featured products for multiplex digital PCR

Publications with dPCR multiplexing

The scientific community has fully embraced the benefits of dPCR multiplex assays. Take a look at a select list of published researchers using the digital PCR multiplexing approach:

Application	Use of multiplex dPCR	References
Investigation of the antimicrobial potential of hydroquinine and changes in gene expression that may contribute to bacterial resistance in P. aeruginosa.	Multiplex reverse transcription digital PCR (mRT-dPCR), for checking the presence of multiple genes associated with efflux pumps	Rattanachak N, Weawsiangsang S, Jongjitvimol T, Baldock RA, Jongjitwimol J. Hydroquinine possesses antibacterial activity, and at half the MIC, induces the overexpression of RND-type efflux pumps using multiplex digital PCR in Pseudomonas aeruginosa. Tropical Medicine and Infectious Diseases. 2022; 7(8):156.
Identification and deconvolution of mixtures of species commonly found in households for forensic purposes	Identified Homo sapiens, canine, feline, bovine swine, pisces, and gallus in two multiplexes	Ghemrawi M and McCord B. Development of a nanoplate-based digital PCR assay for species identification with mixture deconvolution. Forensic Science International: Genetics Supplement Series. 2022; 8:193–195.
Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater	Targeted multiplex dPCR assay for detection of four different Variants of Concern (VOC) and quantification of the RNA from different SARS-CoV-2 VOC in influent wastewater	Booagaerts T et al. Optimization and application of a multiplex digital PCR assay for the detection of SARS-COV-2 variants of concern in Belgian influent wastewater. Viruses. 2022; 14(3):610.
Detection of gene fusions from RNA	Multiplexed primers in initial RT-PCR reaction in ASPYRE technology to identify 37 potential targets within the 3’ gene fusion families	Gray ER at al. Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE. BMC Medical Genomics. 2022; 15:215.

More resources on mutliplex dPCR

Further References

Whale AS, Huggett JF, Tzonev S. Fundamentals of multiplexing with digital PCR. Biomolecular Detection and Quantification. 2016; 10:15–23.
Izadpanah M et al. Simple and fast multiplex PCR method for detection of species origin in meat products. Journal of Food Science and Technology. 2017; 55(2): 698–703.
Lee YM, Lee S, Kim HY. A multiplex PCR assay combined with capillary electrophoresis for the simultaneous identification of Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock. Foods. 2021; 10(11):2631.
Pecoraro S et al. Overview and recommendations for the application of digital PCR. EUR 29673 EN, Publications Office of the European Union, Luxembourg, 2019. https://gmo-crl.jrc.ec.europa.eu/doc/WG-dPCR-Report.pdf.
Dobnik D, Spilsberg B, Bogožalec K, Holst-Jensen A and Žel J. Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction. Analytical Chemistry. 2015; 87(16):8218–8226.
Lao TD, Le TAH. Exploring the multiplex PCR for detection of animal-derived ingredients in vegetarian foods. Pharmacopore. 2020; 3.
Boukharouba A, González A., García-Ferrús M., Ferrús A. Botella S. Simultaneous detection of four main foodborne pathogens in ready-to-eat-food by using a simple and rapid multiplex PCR (mPCR) assay. International Journal of Environmental Research and Public Health. 2022; 19(3): 1031.
Appay R et al. Multiplexed droplet digital PCR assays for the simultaneous screening of major genetic alterations in tumors of the central nervous system. Frontiers in Oncology. 2020; 10:579762.
Peitz C et al. Multiplexed quantification of four neuroblastoma DNA targets in a single droplet digital PCR reaction. Journal of Molecular Diagnostics. 2020; 22(11):1309–1323.
Jiang L et al. Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument. Scientific Reports. 2020; 10: 19892.
Oscorbin I, Kechin A, Boyarskikh U., Filipenko M. Multiplex ddPCR assay for screening copy number variations in BRCA1 gene. Breast Cancer Research and Treatment. 2019; 178(3):545–555.
Hughesman CB et al. A robust protocol for using multiplexed droplet digital PCR to quantify somatic copy number alterations in clinical tissue specimens. PLOS ONE. 2016; 11(8): e0161274
Yu Q. et al. Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients. Molecular Medicine Reports. 2017; 16(2):1157–1166.
Andersen RF And Jakobsen A. Screening for circulating RAS/RAF mutations by multiplex digital PCR. Clinica Chimica Acta. 2016; 458:138–143.
Corné J et al. Development of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients. Scientific Reports. 2021; 11(1):17316.
Dong L et al. A rapid multiplex assay of human malaria parasites by digital PCR. Clinica Chimica Acta. 2023; 539:70–78.
Shi K et al. A multiplex crystal digital PCR for detection of African Swine Fever Virus, Classical Swine Fever Virus, and Porcine Reproductive and Respiratory Syndrome Virus. Frontiers in Veterinary Science. 2022; 9:926881.
Wu, J et al. Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool. Crit Care. 2022; 26: 243.
Zhu X et al. Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections. Virology Journal. 2022; 19:70.
Maggi R, Breitschwerdt EB, Qurollo B, Miller JC. Development of a multiplex droplet digital PCR assay for the detection of Babesia, Bartonella, and Borrelia species. Pathogens 2021; 10(11):1462.
Shin J et al. Duplex dPCR system for rapid identification of gram-negative pathogens in the blood of patients with bloodstream infection: A culture-independent approach. Journal of Microbiology and Biotechnology. 2021; 31(11):1481–1489.
Te SH, Chen EY, Gin KYH. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria Cylindrospermopsis and Microcystis. Applied and Environmental Microbiology. 2015; 81(15):5203–5211.
Hayes D and Dobnik D. Commentary: Multiplex dPCR and SV-AUC are promising assays to robustly monitor the critical quality attribute of AAV drug product integrity. Journal of Pharmaceutical Sciences 111(8): 2143–2148.
Nell RJ et al. Accurate quantification of T Cells in copy number stable and unstable DNA samples using multiplex digital PCR. Journal of Molecular Diagnostics. 2022; 24(1):88–100.
Sanchez MEN et al. Multiplex reverse transcriptase droplet digital PCR for the simultaneous quantification of four dengue serotypes: Proof of concept study.2020; Biologicals 67:62–68.
Meng P et al. Development of a multiplex dPCR assay for ERBB2 amplification in breast cancer. European Journal of Cancer. 2022; 175(1):S80.
Morisset D, Štebih D, Milavec M, Gruden K, Žel J. Quantitative analysis of food and feed samples with droplet digital PCR. PLoS One. 2013; 8(5): e62583.
Oscorbin IP et al. Multiplex droplet digital PCR assay for detection of MET and HER2 genes amplification in non-small cell lung cancer. Cancers (Basel). 2022; 14(6):1458.
Center for Breakthrough Medicines. https://breakthroughmedicines.com/all/tech-note-high-throughput-aav-viral-titering-using-nanoplate-based-digital-pcr/ (Accessed April 21, 2023)