What can you do with digital PCR?
Whether you’re handling precious samples, analysing rare mutations or studying samples fraught with inhibitors, nanoplate dPCR offers precise and reproducible data. The fast, automated workflow substantially reduces variability and improves consistency while being so simple that it requires very little training to master.
Digital PCR, in particular nanoplate dPCR on the QIAcuity, is revolutionizing research by fundamentally changing the questions you can ask and answer. The method fuels applications that were previously hindered by the limitations of qPCR and other dPCR technologies. See what the technology can do for your specific application below.
Rare mutation detection
Rare mutation detection (RMD) refers to detecting a sequence variant that is only present at a very low frequency in a pool of wild-type backgrounds (less than 1% or even 0.1%). Thus, for detecting and quantifying rare events, such as point mutations or single nucleotide polymorphisms (SNPs), a sensitive, accurate and precise method is necessary. The challenge is the discrimination between two highly similar sequences, of which one is significantly more abundant than the other.
Benefits of using nanoplate dPCR for detecting rare mutations
- Ability to load a large input reaction volume into 26,000 partitions, which substantially increases the chances of finding a rare target
- Multiplexing for mutant and wild-type sequencing to detect low fractions of rare mutant molecules against an abundant wild-type background
Explore our QIAcuity solutions for clinical testing and oncology research
Copy number variation analysis
Copy number variation (CNV) analysis determines the number of copies of a particular gene in an individual's genome. It is known that genes occur in two copies per genome; however, these genes can occur more often in some cases. Gene amplification (which activates oncogenes) and deletion (which inactivates tumor suppressor genes) are important copy number alterations (CNAs) that affect cancer-related genes, in addition to the genomic changes such as point mutations, translocations and inversions. Most cancer-related genes affected by CNAs have been defined as critical genes in cancer-signaling pathways involved in carcinogenesis and cancer progression. CNVs are an essential source of genetic diversity (deletion or duplication of a locus) and allow studying genes associated with common neurological and autoimmune diseases, genetic conditions and adverse drug responses.
Benefits of using nanoplate dPCR for CNV analysis
- Detection of less than 1.2-fold change in CNVs, with results in about 2 hours
- Improved economic and throughput level of dPCR CNV analysis with the 8.5K Nanoplate or multiplexing capabilities or finer discrimination of consecutive copy number states with the 26K Nanoplate
- Automatic calculation of CNVs with the QIAcuity Software Suite and possibility to design custom assays
Liquid biopsy
A liquid biopsy, also known as fluid biopsy or fluid-phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. It is mainly used as a diagnostic and monitoring tool for diseases such as cancer. Liquid biopsy is less invasive for the donor compared to tissue biopsy. When tumor cells die, they release ctDNA into the blood. Cancer mutations in ctDNA mirror those found in traditional tumor biopsies, allowing them to be used as molecular biomarkers to track the disease. The challenge is the low concentration of ctDNA from the tumor cells in the blood. The gold standard has been to use NGS, pyrosequencing or real-time qPCR, but the drawback of these methods has been their limitations in LOD. Pyrosequencing for tumor tissue is about 10%, NGS is between 1–5%, and qPCR can detect down to 1%. This creates an issue for relapse during residual disease monitoring of the donor because of the limitation in detection levels.
Benefits of using nanoplate dPCR for liquid biopsy analysis
- Load up to 28 µl sample to increase the LOD and minimize subsampling error with the QIAcuity Nanoplate 26K – allows you to generate more data points to secure small changes in expression or for residual disease monitoring
- Detect ultra-rare mutations down to 0.01% variant allele frequency
- Handle more crude samples like whole blood and urine, as dPCR measurement is unaffected by amplification efficiency
Microbial detection
The increasing prevalence of infectious diseases and epidemic outbreaks underscores the need for improved detection and analysis of microbes, particularly pathogens. The combination of speed, high sensitivity, accuracy, and absolute quantification is essential for both pathogen detection and microbiome analysis in public health and epidemiology. Digital PCR, as a fast, sensitive and precise method, is highly beneficial for identifying, detecting, characterizing and monitoring changes in pathogens and microbiomes. The application areas of dPCR in microbial detection range from pathogens in food, drug resistance, microorganism research, investigating antimicrobial resistance genes and analysis of viral/bacterial-host relationships.
Benefits of nanoplate dPCR for microbial detection
- Precise and absolute quantification of microbial material even from complex samples or samples with high levels of inhibitors
- Larger sample volumes (up to 28 µl) with Nanoplate 26K for boosting sensitivity and detecting targets below the detection limits of other commercial assays
- Multiplexing up to five assays possible with a selection from custom-designed assays or more than 700 catalogue assays for microbial targets (bacterial, viral, virulence factors, AMG, etc.)
Viral load quantification
Viral load testing measures the amount of a specific virus in a biological sample. Results are reported as the number of copies of the viral RNA per milliliter of sample. Viral load tests are used to diagnose acute viral infections, guide treatment choices and monitor response to medical treatment.
Benefits of using nanoplate dPCR for detecting virulence genes
- Detect low-abundance genes with the Nanoplate 26K, which allows more partitioning per sample and a higher sample load volume
- Ability to analyze both microbial and viral targets, with highly specific detection of only the sequence of interest
- Accurate and efficient analysis by multiplexing up to five targets in one reaction
Cell and gene therapy
Digital PCR enables a range of gene therapy applications, including adeno-associated virus vector genome titer, lentiviral vector copy number measurement, and CAR-T cell therapy development and manufacturing. This is critical when developing effective and reproducible cell and gene therapies while ensuring patient safety.
Viral titer measurement
Discover how QIAcuity dPCR can generate the same level of accuracy and precision in viral titer quantification as the traditional ddPCR method at increased speed and overall higher throughput and scalability. Discover a complete workflow from viral vector lysis to quantification of residual DNA to vector genome titration and genome integrity determination with superior accuracy, reproducibility and speed.
Benefits of using nanoplate dPCR for AAV titration
- Consistent and robust determination of final titer thanks to efficient capsid lysis and residual DNA removal with our specialized kits
- Reduced errors and easy implementation with one protocol with minimal number of manual steps and only 10 minutes of hands-on time
- Higher accuracy and flexibility thanks to multiplexing with as many as 10 single target assays with different dye combinations; option to extend to 5-plex capacity with genes of interest (GOI) assays
- Precise assessment of genome integrity using up to 5 targets simultaneously enabled by our advanced QIAcuity software feature
Residual host cell DNA quantification
Residual host cell DNA (HCD) is carried over during the manufacturing processes of biopharmaceutical products. The acceptable levels are established by regulatory agencies such as the U.S. Food and Drug Administration and the World Health Organization. Digital residual DNA quantification kits are ideal for the highly precise quantification of HCD in complex bioprocesses. Common host cells used during the development of gene therapies, therapeutic proteins and other biotherapeutics include Human Embryonic Kidney 293 (HEK293), Chinse Hamster Ovary (CHO), and E. coli.
Benefits of using nanoplate dPCR for residual host cell DNA quantification
- Easy setup and detection of host cell DNA thanks to a premixed master mix and a positive/internal control
- Accurate detection of E. coli, CHO and HEK293 residual DNA to the low femtograms, even in the presence of PCR contaminants and inhibitors
- Multicopy species-specific target assays ensure results are unaffected by the fragmentation level of the resDNA
- Validation of quantitation accuracy or bridging studies possible using the dPCR-verified standards
Mycoplasma detection
Mycoplasmas are contaminants of biological products derived from cell lines in the biopharmaceutical industry. Mycoplasmas can appear in cell culture as a result of contamination of the source cell lines themselves (cell substrates) or from adventitious introduction of mycoplasmas during production. Multiple contamination risk guidelines and technical papers on mycoplasma safety for the manufacturing of biological products are available.
Digital PCR can be used to detect contamination in cell cultures and other cell culture-derived biologicals. For example, the QIAcuity Mycoplasma Quant Kit is an RT-dPCR kit that detects rRNA and DNA, allowing for high sensitivity of the method. An internal amplification control prevents false negatives due to PCR inhibitors, improper RNA extraction or improper RT reaction. The probe-based assay can quantify and detect 127 different mycoplasma species.
Benefits of using nanoplate dPCR for mycoplasma detection
- Compliant: NAT (Nucleic Acid Technique) workflow for mycoplasma testing that is compliant with the European, US and Japanese Pharmacopeia
- Fast: No time-consuming cultivation of mycoplasma necessary
- Sensitive: Detection of rRNA enables a higher sensitivity than using only DNA because of the multiple copies present in a single bacterial cell (detection of < 10 CFU/mL). The assay is still able to detect DNA if the RT-step is skipped, enabling a high degree of flexibility.
- Pre-validated: The workflow has been extensively tested as part of a comprehensive validation report that can reduce your own validation efforts
- Ten Mycoplasma Standard CFU Kits for in-house validation or as positive control without introducing vital mycoplasma
Gene expression analysis
Gene expression profiling simultaneously compares the expression levels of multiple genes between two or more samples. This analysis can help scientists establish the molecular basis of phenotypic differences and select gene targets for in-depth study. Gene expression profiling provides valuable insight into the role of differential gene expression in normal biological states and diseases.
Benefits of nanoplate dPCR for gene expression quantification
- Detect small-fold changes, especially in low-template amounts
- Validate results with an absolute concentration and abundance below 1% depending on the input amount
- Achieve high precision and high dynamic range of log5 with Nanoplate 26K or perform economical runs with 12 µl reactions and high-throughput options for “similar” expressed targets (up to about 4-fold expression change) on a Nanoplate 8.5K
miRNA expression analysis
MicroRNA (miRNA) expression profiling simultaneously compares the expression levels of multiple or single miRNAs between two or more samples. This analysis can help scientists identify and quantify miRNA as a biomarker in diseases such as cancer. It provides valuable insight into the role of miRNA expression in normal biological states and diseases.
Benefits of nanoplate dPCR for miRNA expression analysis
- Discrimination of single nucleotide differences in closely sequence-related miRNAs thanks to the high specificity of dPCR
- Absolute quantification of subtle miRNA expression changes, especially in low template amounts
Food testing
Multiplex digital PCR assays have a wide range of applications in the food industry, including regulatory control, quality assurance, GMO testing, food fraud detection, and foodborne disease monitoring. These assays can identify animal species and trace the origin of meat products, such as distinguishing between pig, camel, sheep, donkey, goat, cow, and chicken in a single reaction. They are also used for quantifying transgenes in GMO testing, with studies showing higher sensitivity and repeatability compared to qPCR. For detecting food fraud, dPCR assays can identify animal-derived ingredients in vegetarian or vegan products by targeting specific mitochondrial and chloroplast DNA markers. Additionally, dPCR is effective in simultaneously detecting multiple microbial pathogens, such as E. coli, L. monocytogenes, S. aureus, and S. enterica, ensuring food safety and quality.
Benefits of using nanoplate dPCR for food testing
- Ideal for complex matrix samples thanks to multiplexing and the ability of dPCR to minimize the effect of amplification efficiencies caused by matrix differences between reference materials and samples
- High-throughput possibilities thanks to multiplexing of up to five channels and an 8-plate system
- Reliable detection of rare GMO events due to reduced background by partitioning
Single-cell analysis
Compared to traditional methods of analyzing cell populations in bulk, single-cell analysis can obtain data at the single-cell level, helping researchers better understand cellular heterogeneity, biological functions, processes and disease mechanisms. Commonly used methods for single-cell analysis, including PCR, qPCR or next-generation sequencing (NGS) sometimes lack the sensitivity required to detect the target of interest.
Digital PCR is an emerging option for single-cell analysis, due to cost-efficient, intuitive and accurate dPCR platforms that offer high throughput, high sensitivity of detection and precision.
Benefits of using nanoplate dPCR for single-cell analysis
- Highly accurate with physical partitioning that is more stable than droplets
- Probe-based detection allows for multiplexing of up to five targets in a single dPCR reaction with minimal optimization
- Precise results enable analysis of low abundance targets and multi-copy targets at a single-cell level
Genome edit detection (CRISPR-Cas9)
Nucleases such as zinc-finger (ZFN), transcription activator-like effector (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR) are used to edit the genome of any cell. These nucleases produce site-specific DNA double-strand breaks (DSBs), which then can be repaired by imprecise, error-prone non-homologous end joining (NHEJ) (donor template/precise point mutation) or by homology-directed repair (HDR) (deletion/indels/insertions) pathways leading to targeted mutagenesis. As a result, a mixed population of cells with heterogeneous indel errors and varying allelic editing frequencies develop. Then, genome editing frequencies at the desired locus are measured. Clonal cell lines isolate single cells, which are then assayed to verify the genome editing event.
Benefits of using nanoplate dPCR for genome edit detection (CRISPR-Cas9)
- Higher sensitivity enabling detection of editing events present at frequencies of 0.5%
- Absolute quantification of editing events from as little as 5 ng of total gDNA
- Ability to distinguish between homozygous and heterozygous edits in clonal populations
NGS library quantification
Next-generation sequencing library quantification is an essential step for using your flow cells at full efficiency. Evidence shows that overloading or underloading an NGS library following inaccurate library quantification adversely affects data output and quality. Using digital PCR to determine the absolute concentration of an NGS library pool can be greatly beneficial for obtaining optimal yield and reducing cost per sample.
Benefits of using nanoplate digital PCR for NGS library quantification
- Absolute quantification of amplifiable library fragments without amplification bias and without standard bias with results in 2 hours, suitable for routine testing
- High reproducibility and superior uniformity for library pooling with coverage of all Illumina library types with one assay
Protein quantification and interaction
Actome’s Protein Interaction Coupling (PICO) technology benefits from the QIAcuity Digital PCR System to offer a highly versatile and sensitive approach for detecting and quantifying single proteins and protein interactions. The PICO technology translates complex protein status into DNA barcodes that can be amplified and detected using dPCR. This is particularly useful when investigating cellular pathways, looking for protein biomarkers, developing novel assays for pharmaceutical research or performing multi-omics analysis.
Benefits of using nanoplate dPCR for protein detection
- The only technology on the market for quantifying proteins using dPCR
- Detect proteins, protein-protein interaction and post-translational modifications at single-cell level
