Applications of digital PCR

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.

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.

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, investigating antimicrobial resistance genes, etc. In pathogen detection, microbial pathogens are often detected simultaneously with viruses, such as in the viral/bacterial-host relationship.

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.

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.

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.

Publications

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

• Huggett JF et al. (2022) Monkeypox: another test for PCR. Euro Surveill., 27(32).
• Amman, F et al. (2022) Viral variant-resolved wastewater surveillance of SARS-CoV-2 at national scale. Nat Biotechnol.
• Tasnim T et al. (2022) A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L in Tulsi churna. Food Control. Volume 137, 108790.
• Alexandra S et al. (2022) Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics. Methods. Volume 201, Pages 5-14.
• Nyaruaba R et al. (2022) Digital PCR applications in the SARS-CoV-2/COVID-19 era: a roadmap for future outbreaks. Clin Microbiol Rev. Sep 21;35(3):e0016821.
• Sun N et al. (2022) Coupling lipid labeling and click chemistry enables isolation of extracellular vesicles for noninvasive detection of oncogenic gene alterations. Adv. Sci., 9, 2105853.
• Piao XM et al. (2022) Expression of RPL9 predicts the recurrence of non-muscle invasive bladder cancer with BCG therapy. Urol Oncol. May;40(5):197.e1-197.e9.
• Zaytseva M et al. (2022) Methodological challenges of digital PCR detection of the histone H3 K27M somatic variant in cerebrospinal fluid. Pathol Oncol Res. Apr 12;28:1610024. 
• Christoph S et al. (2022) Effects of varying flux and transmembrane pressure conditions during ceramic ultrafiltration on the infectivity and retention of MS2 bacteriophages. Separation and Purification Technology. Volume 299, 121709.
• Lindsey AM et al. (2022) Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products. Cytotherapy.
• Boogaerts T et al. (2021). An alternative approach for bioanalytical assay optimization for wastewater-based epidemiology of SARS-CoV-2. Science of The Total Environment, Volume 789, Article 148043.
• Luo Y et al. (2020). Massively parallel single-molecule telomere length measurement with digital real-time PCR. Sci Adv. 6(34):eabb7944.
• Lee EG et al. (2021). TOMM40 RNA Transcription in Alzheimer’s Disease Brain and Its Implication in Mitochondrial Dysfunction. Genes. 12, 871.
• Wirtz RM et al. (2021). FGFR testing from matched tissue and urine samples within the prospective real world clinico-pathological register trial BRIDGister. Journal of Clinical Oncology. 39:15_suppl, e16532-e16532.
• [Preprint version only] Yang JX et al. (2021). Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes.
• Ferasin L et al. (2021). Infection with SARS-CoV-2 variant B.1.1.7 detected in a group of dogs and cats with suspected myocarditis. Vet Rec. 2021 Nov;189(9):e944.
• [Preprint version only] Wu X et al. (2021). A Warm-start Digital CRISPR-based Method for the Quantitative Detection of Nucleic Acids.
• [Preprint version only] Viveros ML et al. (2021). Wild Type and Variants of Sars-Cov-2 in Parisian Sewage: Dynamics in Raw Water and Fate in Wastewater Treatment Plants.
• Romanelli K et al. (2021) Clinical and molecular characterization of thyroid cancer when seen as a second malignant neoplasm. Therapeutic Advances in Endocrinology and Metabolism. Volume: 12
• Murphy LA et al. (2021) Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR. Blood. 138 (Supplement 1): 4001.
• [Preprint version only] Toffoli M et al. (2021) Comprehensive analysis of GBA using a novel algorithm for Illumina whole-genome sequence data or targeted Nanopore sequencing.
• Passera A et al. (2021) Bacterial Communities in the Embryo of Maize Landraces: Relation with Susceptibility to Fusarium Ear Rot. Microorganisms, 9(11), 2388.