Applications of digital PCR

What can you do with digital PCR?

Digital PCR and in particular the QIAGEN nanoplate-based technology is revolutionizing research by fundamentally changing the questions you can ask and answer today, fuelling applications that were previously hindered by the limitations of qPCR and other dPCR technologies. The following section describes the benefits of using digital PCR in some of the current and emerging applications.

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.

An example of rare mutation detection is detecting a low-frequency single nucleotide mutation in a cancer biopsy sample.

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 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.

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.

Quantifying Wolbachia-Nasonia interactions
This work compares the performance of qPCR vs. dPCR in the quantification of gene expression and Wolbachia abundances in Nasonia parasitoid wasps.

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 acute diseases such as cancer. It provides valuable insight into the role of miRNA expression in normal biological states and diseases.

Microbial pathogen detection

The combination of speed, high sensitivity, accuracy, and absolute quantification is essential for both pathogen detection and microbiome analysis in public health and epidemiology. It is imperative when studying phylogeny for identification, detection, characterization and monitoring of changes in pathogens and microbiomes. The application area is broad, ranging from pathogens in food, drug resistance, microorganism research, etc. In pathogen detection, microbial pathogens are often detected simultaneously with viruses, such as in the viral/bacterial-host relationship.

Vector-borne diseases identification and quantification
The results presented in this QIAcuity dPCR vs. qPCR comparison study demonstrate precise and absolute detection and quantitation of low abundant vector-borne viruses in mosquitoes.
Wastewater monitoring with dPCR

Screening wastewater, or sewage, for the presence of pathogens can be an effective surveillance method. Because natural sewage is highly heterogeneous and qPCR data can be highly variable due to inadequate sample dilution or chemical contamination, a method capable of identifying such rare target nucleic acid molecules from a mixture of non-target backgrounds is required. Absolute quantification with dPCR facilitates accurate and sensitive detection of these rare molecules from wastewater samples because it overcomes the problem of variability, reduces the impact of PCR inhibitors, and eliminates the need for standard curves.

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.

Digital PCR for SARS-CoV-2 detection in human saliva
Read an application note describing the use of QIAcuity digital PCR as an alternative to qPCR for precise, more sensitive and high-frequency detection of low levels of the virus and its variants in non-invasive saliva specimens.

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.

cfDNA mutation analysis by dPCR
The article introduces the power of two: a workflow that starts with high-volume sample processing and ends with ultra-low mutation detection to produce reliable results.

GMO detection 

Genetically modified organism (GMO) is commonly used to refer to genetically altered crops. Genetic engineering provides the technology to introduce certain desired traits, such as virus/insect resistance, increased crop yields, enhanced composition, etc. GMO detection can be qualitative (presence) or quantitative (amount of GMO). It can either be event-specific, that detects the presence of a DNA sequence unique to the specific GMO or construct-specific, that detects a foreign DNA sequence inserted in a GMO. GMO testing is necessary for many crop producers/exporters/importers to meet regulatory requirements. Real-time PCR, which is currently the go-to method, is limited in detecting and quantifying low DNA targets, often seen in complex food/feed matrices. 

Genome edit detection (CRISPR-Cas9)

In genome editing studies, 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.

NGS library quantification and validation

NGS library quantification and validation are performed with different methods today. The use of spectrophotometric and fluorometric systems and qPCR is limited in accurately quantifying generated libraries. Having an accurate concentration of your libraries is crucial for a cost-effective and accurate sequencing run. Real-time PCR has so far been the gold standard for validation of the sequencing run. The drawback is the lack of precision when you need to validate findings in your NGS below 1%. 

Residual host cell DNA quantification

Residual host cell DNA (HCD) is carried over during the manufacturing processes of therapeutic proteins and vaccines. 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. Cells that undergo clearance during the development of gene therapies, cell-based vaccines and similar biotherapeutics or others could be HEK293, CHO or E. coli.

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Publications

Browse a growing list of articles featuring applications using the QIAGEN nanoplate dPCR technology.