Nanoplate digital PCR publication roundup

Nanoplate dPCR technology – the basis of the QIAcuity Digital PCR System – is gaining attention by enabling a vast scope of research applications. Researchers globally are conveying their breakthrough findings, latest discoveries and developments to a growing catalog of published studies serving as a strong testament to the accessibility of this technology. The number of publications citing QIAcuity dPCR just three years after its launch is over 200 – a number that is steadily growing compared to the ddPCR method when it was introduced over a decade ago.

Here’s a rundown of a few interesting studies that used nanoplate dPCR technology, demonstrating its relevance and potential in key application areas, including oncology, cell therapy, food sciences, viral detection, and mutation detection.

Oncology

Study: Clinical and molecular characterization of thyroid cancer when seen as a second malignant neoplasm

What it looked at: The study’s objective was to characterize clinical and pathological findings of patients with thyroid cancers that developed as second malignant neoplasms and investigate genetic alterations in thyroid cancer tissue samples.

How dPCR helped: The researchers identified BRAFV600E mutations and RET/PTC fusions by performing microfluidic dPCR procedures using QIAcuity dPCR.

Significance: dPCR facilitates precise and sensitive detection of gene mutations and fusions in thyroid cancer tissue samples. You can read about the study here.

Study: FGFR testing from matched tissue and urine samples within the prospective real-world clinico-pathological register trial BRIDGister

What it looked at: The study aimed to examine fibroblast growth factor receptor (FGFR) alterations in urine and tissue samples from patients suspected to have bladder cancer and undergoing their first transurethral resection biopsy.

How dPCR helped: The researchers centrally analyzed urine samples using QIAcuity dPCR.
Significance: dPCR provides outstanding sensitivity for testing FGFR mutations and fusions in urine samples. You can read the study here.

Study: Expression of RPL9 predicts the recurrence of non-muscle invasive bladder cancer with BCG therapy

What it looked at: The study aimed to identify a novel marker that more accurately predicts the prognosis of patients with primary or recurrent non-muscle invasive bladder cancer (NMIBC) receiving Bacillus Calmette-Guerin (BCG) therapy.

How dPCR helped: The researchers used dPCR to evaluate patients with NMIBC. The dPCR analysis revealed the downregulation of RPL9 in patients with recurrence after BCG therapy.

Significance: dPCR identifies the expression of RPL9 in patients with NMIBC and supports accurate prognosis prediction in clinical settings. You can read more about the study here.

Study: Methodological challenges of digital PCR detection of the histone H3 K27M somatic variant in cerebrospinal fluid

What it looked at: The study aimed to develop a reliable cross-platform dPCR method for detecting H3 K27M variant in cell-free DNA (cfDNA) isolated from cerebrospinal fluid (CSF) of pediatric patients with central nervous system (CNS) tumors.

How dPCR helped: The researchers used dPCR technology to detect the histone H3 K27M mutation in cerebrospinal fluid (CSF).

Significance: dPCR protocol enables the detection of mutations with extremely low allele burden, as observed in the detection of H3 K27M somatic variant in CSF of pediatric patients with CNS tumors with superior sensitivity. You can read about the study here.

Study: Coupling lipid labeling and click chemistry enables isolation of extracellular vesicles for noninvasive detection of oncogenic gene alterations

What it looked at: The study aimed to quantitatively detect gene alterations, i.e., EWS/FLI-1 rearrangements in Ewing sarcoma and KRAS mutations in pancreatic cancer. After conducting multifaceted optimization of the experimental conditions, the authors established a streamlined workflow that integrates Click Beads-based extracellular vesicles (EV) capture and reverse transcription digital polymerase chain reaction (RT-dPCR) quantification of EV-derived mRNA.

How dPCR helped: The researchers subjected EV-derived mRNA to downstream analysis by RT-dPCR to obtain absolute quantification of cancer-specific gene alterations.

Significance: The Click Bead-based EV capture method combined with RT-dPCR-based quantification of oncogenic gene alterations provides a noninvasive diagnostic solution with potential clinical applications in detecting disease progression and monitoring treatment responses for Ewing sarcoma or pancreatic cancer patients. You can read about the study here.

Cell therapy

cell and gene editing, genetic editing and gene research, in vitro

Study: Detection of vector copy number in bicistronic CD19xCD22 CAR T cell products with digital PCR

What it looked at: Motivated by the drawbacks of quantitative PCR (qPCR) in vector copy number (VCN) assessment in CAR T cell products, the study observed dPCR techniques for detecting and quantifying VCN in Bicistronic CD19xCD22 CAR T cell products.

How dPCR helped: The researchers used dPCR to analyze vector copy number in Bicistronic CD19xCD22 CAR T cell products.

Significance: dPCR methods can be adopted for absolute quantification of CAR transgenes and precise and accurate VCN estimation. You can read about the study here.

Study: Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products

What it looked at: Inspired by the challenges of measuring VCN in CAR T-cell products with qPCR, the study aimed to compare dPCR techniques with qPCR in detecting and quantifying CAR transgenes.

How dPCR helped: The researchers developed a dPCR assay to analyze the novel bicistronic CD19 × CD22 CAR T-cell construct.

Significance: dPCR supports a more precise analysis and quantification of CAR transgenes and VCN measurement compared with traditional PCR methods with better test-retest variability. You can read about the study here.

Food sciences

Applied testing, food safety testing, sample collection from salami

Study: A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L. in Tulsi churna

What it looked at: The study’s goal was to develop a simple and cost-effective PCR method for identifying two plant species, Ocimum basilicum, and Ocimum tenuiflorum, found in a herbal product called Tulsi churna.

How dPCR helped: The researchers developed a dPCR assay to detect both plant species simultaneously.

Significance: dPCR assays are highly suitable and sensitive methods for detecting and retrieving plant species in herbal products. You can read more about the study here.

Viral detection

Study: Digital PCR can augment the interpretation of RT-qPCR Cq values for SARS-CoV-2 diagnostics

What it looked at: A secondary objective of the study was to examine the impact of dPCR as a confirmatory tool for developing SARS-CoV-2 molecular diagnostic tests.

How dPCR helped: The study used dPCR to characterize controls to be used in SARS-CoV-2 diagnostic tests.

Significance: The study highlights that RT-dPCR shows impressive potential for characterizing controls during assay development, hence playing a significant role in determining the efficacy of routine SARS-CoV-2 molecular diagnostic tests. You can read the study here.

Study: Digital PCR applications in the SARS-CoV-2/COVID-19 era: a roadmap for future outbreaks

What it looked at: Driven by the pitfalls of RT-qPCR for diagnosing patients with SARS-CoV-2, the study reviewed select publications to highlight the impact of dPCR in applications, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2. The study also compared its performance to RT-qPCR for rapid and accurate diagnosis of SARS-CoV-2.

How dPCR helped: The study reviewed how dPCR has been used in various applications to solve SARS-CoV-2 pandemic-related problems.

Significance: The study highlights that RT-dPCR is superior to RT-qPCR in terms of specificity, sensitivity, reproducibility, and detection limits for SARS-CoV-2. This superiority is visible in low-viral-load samples. It also demonstrates that dPCR is a promising tool for diagnosing and monitoring emerging or reemerging infectious diseases. You can read more about the study here.

Study: Effects of varying flux and transmembrane pressure conditions during ceramic ultrafiltration on the infectivity and retention of MS2 bacteriophages

What it looked at: The study investigated the effects of varying flux and transmembrane pressure conditions during ceramic ultrafiltration on the infectivity and retention of MS2 bacteriophages.

How dPCR helped: The researchers used dPCR to measure the ratio of plaque forming units (PFU) indicating infectious MS2 phages and the total amounts of infectious and non-infectious MS2 bacteriophages.

Significance: dPCR enables absolute quantification of infectious and non-infectious viruses like MS2. You can read the study here.

Transplantation

Study: New methods for the quantification of mixed chimerism in transplantation

What it looked at: The study investigated new technologies such as dPCR and NGS for the accurate quantification of chimerism in the context of hematopoietic stem cell transplantation (HSCT) and compared them against traditional methods.

How dPCR helped: The researchers used new dPCR and NGS methods that increased the sensitivity of chimerism quantification, allowing microchimerism (<1%) detection in HSCT. dPCR using the QIAcuity enabled precise, reliable and high-sensitivity detection of chimerism after transplantation. It allowed high-order genotyping marker multiplexing amplification and automated the dPCR steps in the same instrument, reducing run time and manual steps that are a source of error. Furthermore, it allowed a homogeneous partitioning in volume that is reproducible in number, which is critical for integrating this technology in diagnostic laboratories.

Significance: These new technologies of dPCR and NGS can allow the combined analysis of chimerism from genomic and cell-free DNA, which is the future of organ transplantation monitoring. You can read the study here.

Key takeaway

Despite the fact that qPCR is still the standard tool for nucleic acid quantification, researchers are beginning to explore and appreciate the potential of dPCR as a superior quantification technology that can power applications previously unimaginable.

You can learn more about how nanoplate dPCR can support your research endeavors here.

References

Spotlight on nanoplate dPCR applications: A growing catalog of published research