Digital PCR

Nanoplate dPCR applications

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 (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

A true needle in a haystack problem

Detection of low-frequency mutations in liquid biopsies is like finding a needle in a haystack. Digital PCR can detect and quantify these rare molecules and offer new opportunities for specific biomarker discovery, early tumor detection and monitoring treatment response. The QIAcuity digital PCR in nanoplates, with its walkaway workflow, superior partitioning and a unique hyperwell feature, extends the exquisite sensitivity and precision of a dPCR method to finding that one copy of the mutant even in the most challenging samples.

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

Multiplex digital PCR for mitochondrial and genomic target copy number analysis

Explore a workflow for high-throughput analyses of target copy numbers in cultured cells. The process combines fast and accurate cell sorting from CellenONE to ensure an exact number of cells are used in downstream reactions. The samples are subjected to probe-based detection on the QIAcuity Digital PCR System with multiplexing of up to five targets in a single dPCR reaction with minimal optimization. The workflow achieves accurate absolute DNA quantification by dPCR for analysis of low abundance targets as well as multi-copy targets at single-cell level.

Copy variation

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

Liquid biopsy-based detection of PIK3CA mutations from cfDNA using dPCR

In this application note, we demonstrate the utility of the QIAcuity Digital PCR System to confidently detect ultra-rare PIK3CA variants in cfDNA. The manual QIAamp workflows and the automated EZ2 and QIAsymphony workflows delivered cfDNA with high yield and purity from large plasma volumes of up to 10 mL. The automated QIAGEN workflows also eliminated the need for manual pre-enrichment or preparation of plates. Additionally, we demonstrated the comparability of dPCR and Qubit quantifications and the ability of dPCR to quantify PIK3CA mutation frequencies in cfDNA with high precision, cost and time-efficiency. 

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.)

Wastewater monitoring with dPCR

Screening wastewater, or sewage, for pathogens, such as  SARS-CoV-2, Legionella spp. or Salmonellacan predict disease outbreaks and provide essential epidemiological data. However, wastewater is highly heterogenous and a method capable of identifying rare targets in such mixed material is required. This is where the true power of dPCR shines. Absolute quantification with dPCR detects rare events, reduces the impact of PCR inhibitors and eliminates the need for standard curves, simplifying pathogen detection in wastewater.

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

Using dPCR to identify and quantify vector-borne diseases vectored by mosquitos

The QIAcuity Digital PCR System was used to detect and quantify high and low-concentration viruses vectored by mosquitoes that carry West Nile virus ( DMA00455) and Flanders virus in a side-by-side comparison with quantitative real-time PCR (qPCR). The QIAcuity Digital PCR System combined with the QIAcuity One-Step Viral RT-PCR Kit enabled precise detection and quantitation of vector-borne viruses in mosquitoes with more reliable results than qPCR, especially for low-abundance targets. Multiplexing allowed the detection and quantitation of multiple targets in a single reaction, thereby increasing sample throughput at a reduced cost per target.

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

All of our most valuable cell and gene therapy content in one place

Explore our specialized content hub, which demonstrates how using dPCR in cell and gene therapy applications can provide specific advantages: accuracy, speed and convenience. Find the latest application notes, scientific posters, webinars and videos on how dPCR can help you get advanced therapies to the market faster. Discover the many uses of dPCR in your cell and gene therapy projects, from research to bioprocessing to quality control (QC).

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

Direct quantification of residual host cell DNA using the QIAcuity Digital PCR Platform

Residual host cell DNA (resDNA or HCD) monitoring is an important step in the process of manufacturing proteins, vaccines and other biopharmaceuticals. The potential carryover of HCD poses a safety concern and is carefully monitored by regulatory agencies. In this scientific poster, discover how digital PCR has emerged as the method of choice for resDNA quantification due to its unmatched sensitivity and accuracy of detection at a low template input range in complex bioprocess intermediates.

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 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

Comparing dPCR and qPCR for quantifying gene expression and bacterial abundances in wasps

Methods using qPCR for assessing gene expression and bacterial counts in arthropods already exist. Although qPCR is a beneficial method for gene expression analysis, the method suffers from limitations including the need for reference materials. In this application note, compare the performance. In this application note, compare the performance of qPCR and dPCR in the quantification of gene expression and Wolbachia abundances in Nasonia parasitoid wasps.

gene 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

Analysis of miRNAs in defined cell pools and single cells using digital PCR

In this application note, read about a combination of both cellenONE and QIAcuity technology for high-throughput absolute quantification of miRNAs in well-defined cell pools and on a single-cell level. Learn how FastLane lysis buffers reduced hands-on time and the EG-based chemistry of QIAcuity allowed for miRNA analysis without major optimization. Explore the entire workflow from cell sorting with cellenONE technology to miRNA quantification with the QIAcuity to achieve sensitive, reproducible and linear quantification from cell lysates as RT and PCR input. 

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

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

Multiplex digital PCR for analysis of gene expression at a single-cell level

Single-cell gene expression analysis allows us to capture individual cell heterogeneity rather than the average output of a cell population. Transcriptome analysis using common PCR- and NGS-based techniques often lacks the sensitivity required to detect low-abundance single-cell targets. Digital PCR is the go-to method for absolute quantification of RNA targets, enabling subtle changes in the expression of target genes to be studied down to a single-cell level. In this application note, discover a workflow that combines high-accuracy single-cell isolation with nanoplate-based dPCR for high-throughput analysis of gene expression in cultured cells.

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

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

Webinar:  NGS library quantification using nanoplate digital PCR

Next-generation sequencing can be a long, expensive process if the NGS flow cells are used at less than full capacity. The most common cause of loading problems is poor library quantification. In this webinar, learn how to use digital PCR to accurately measure NGS libraries, regardless of their average fragment length and composition.

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

How to measure proteins and protein interactions in biological samples with single molecular sensitivity using protein interaction coupling (PICO) and dPCR

Discover more about the PICO technology, the complete workflow and where QIAcuity digital PCR fits in, the advantages of the PICO approach over Western blots and co-immunoprecipitation and more.

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