Digital PCR Assay Research Articles

Clinical Implementation of a Noninvasive, Multi-Analyte Droplet Digital PCR Test to Screen for Androgen Receptor Alterations

Recent studies have shown that alterations of the androgen receptor (AR) are associated with resistance to AR-directed therapy in prostate cancer. Thus, it is crucial to develop robust detection methods for AR alterations as predictive biomarkers to enable applicability in clinical practice. We designed and validated five multiplex droplet digital PCR (ddPCR) assays for reliable detection of 12 AR targets including AR amplification, AR-V7 and 10 AR hotspot mutations as well as AR and KLK3 gene expression from plasma derived cell-free (cf)DNA and cfRNA.The assays demonstrated excellent analytical sensitivity and specificity ranging from 95% to 100% (95% CI: 75-100%). Intra- and inter-run variation analyses revealed a high level of repeatability and reproducibility. The developed assays were further applied in peripheral blood samples from 77 patients with advanced prostate cancer to assess their feasibility in a real-world scenario. Optimizing the reverse transcription of RNA increased the yield of plasma-derived cfRNA by 30-fold. Among 23 patients with castration-resistant prostate cancer (CRPC), 6 patients (26.1%) had one or a combination of several AR alterations, while only 2 out of 54 patients (3.7%) in the hormone-sensitive stage showed AR alterations. These findings were consistent with other studies and suggest that implementation of a comprehensive AR status detection in clinical practice is feasible and can support the treatment decision-making process.

Abstract 6957: Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary

Abstract The extracellular vesicle (EV) route is essential for cell-to-cell communication. Cancer cells release EVs in the extracellular space, where they can interact with recipient cells inducing regulation of gene expression, activation of specific signaling pathways and tumor microenvironment remodeling. We investigated the EVs released by two cell lines of cancer of unknown primary site (CUP) that we established from patient’s tumor cells, namely CUP#55 and CUP#96, as described in https://www.medrxiv.org/content/10.1101/2023.03.12.23287041v1). CUP comprises 3-5% of new cancer cases and presents with metastases of unknown or uncertain origin and no apparent primary tumor. Our CUP cell lines are both characterized by FGFR2 gene amplification. It has been recognized that tumors use circular extrachromosomal DNA (ecDNA) as a way to increase oncogenic amplification. We hypothesized that FGFR2 amplification in CUP cell lines could be associated with ecDNA generation and that this ecDNA could be loaded as cargo inside EVs. FGFR2 copy number was quantified using a probe-based droplet digital PCR assay then we identified the different nature of FGFR2 amplification in the two models using FGFR2 FISH assay: CUP#55 presented a chromosomal homogeneously staining region while CUP#96 displayed double minute chromosomes. FGFR2 DNA was detectable in the cytoplasm fraction, therefore we isolated extracellular vesicles from culture medium using an ultracentrifugation-based protocol and confirmed the presence of the entire length of FGFR2 gene inside small and large vesicles. Since ecDNA contributes to cancer genome remodeling also forming chimeric circles, we confirmed the circular nature of a fraction of FGFR2 ecDNA using the Plasmid-Safe ATP-dependent DNAse test. Finally, we tested the oncogene delivery capability of CUP EVs by administering CUP#96 and CUP#55 small and large vesicles to recipient cell lines. We observed an increase of FGFR2 DNA at 24 hours upon EV treatments, and its functional transcription in FGFR2 mRNA after 48 hours. In conclusion, we identified a model of oncogene amplification and delivery in two cell lines of cancer of unknown primary, which could explain the high metastatic potential of this cancer type. (The research leading to these results has received funding from AIRC under IG 2021 - ID. 25789 project - P.I. Ferracin Manuela) Citation Format: Irene Salamon, Gianluca Storci, Beatrice Fontana, Salvatore Serravalle, Giulia Gallerani, Roberta Roncarati, Spartaco Santi, Massimiliano Bonafè, Manuela Ferracin. Extrachromosomal FGFR2 is delivered in extracellular vesicles in two models of cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6957.

Abstract 7009: Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR

Abstract DNA methylation is a common epigenetic modification, characterized by the presence of the signature 5-methylcytosine without altering the sequence of DNA. The study of DNA methylation in mammals has gained significant attention due to its broad impact on numerous biological processes and its critical role in the onset and progression of many diseases, such as cancer and aging. Consequently, there is a pressing need for a rapid and precise method to assess methylation status. Currently, most approaches for quantifying DNA methylation rely on sodium bisulfite treatment. However, such approaches do not align with the uracil DNA glycosylase PCR system. In this study, we demonstrate the application of methylation-sensitive restriction enzymes (MSRE) and digital PCR to determine the methylation status of the Ras association domain family 1 isoform A (RASSF1A) in cancer cell lines. Digital PCR enables the precise detection and absolute quantification without the reliance on reference standards. We developed a multiplex digital PCR assay that includes the target gene RASSF1A, along with two reference genes for the purpose of monitoring digestion completion and correcting DNA input. Each sample of interest was measured both before and after a MSRE digestion. To ensure the quantification accuracy, we employed the commercially available CpGenome human methylated DNA standard with a known concentration from MilliporeSigma, treating it with and without a MSRE, and subsequently performing digital PCR experiments using Roche Digital LightCycler® IVD system with a Universal nanowell plate featuring 28,000 partitions. The expected concentrations lay within the 95% confidence interval, affirming the accurate quantification achieved through digital PCR. Next, we measured the fraction of methylated alleles in various lung cancer and breast cancer cell lines, as well as in healthy human gDNA. The methylation of RASSF1A promoter in healthy human gDNA is ~0.7%, while a broad range of methylation levels was observed in cancer cell lines, ranging from ~0.7% to ~100.2%. Notably, the influence of GC bias was identified in this study. To overcome this critical challenge, we optimized the usage of several high GC enhancers. The results demonstrated that the concentration of 5-7.5% DMSO, 7.5% glycerol, and commercially available OneTaq or Q5 GC enhancers from New England Biolabs were effectively incorporated into the Roche digital PCR system, thereby enhancing the accuracy of quantification from 30% to 100%. Together, we demonstrated a remarkable approach for DNA methylation analysis using Roche digital PCR system in combination with MSREs and suitable high GC enhancers. This study suggests a promising rapid, accurate and cost-effective tool to advance research in the field. *Data on file at Roche Diagnostics, Wilmington, MA, USA Citation Format: Yu Zhao, Lindsey Cambria. Quantification of DNA methylation using methylation-sensitive restriction enzymes and digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7009.

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become “digitalized”, which was deemed important to guide clinicians in treatment decision for better patient care.

Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots.

DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high‑throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation‑Sensitive Restriction Enzyme‑droplet digital PCR (MSRE‑ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high‑sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE‑ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE‑ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.

Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues

BackgroundThe disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues.MethodsTo answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes.ResultsIn summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range.ConclusionsOur results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

Abstract B019: Assessing Circulating TumOur DNA as a prognostic biomarker in endometrial cancer (The CODEC Study)

Abstract Introduction/Background: Endometrial cancer (EC) is the most common gynecological cancer in the developing world. While significant progress has been made through molecular sub-classification, the treatment of EC for many women remains contentious and better biomarkers are required to guide therapy selection. Circulating tumor DNA (ctDNA) a ‘liquid biopsy’, involves collection of tumor specific DNA via a blood test and can quantify cancer burden and detect tumor specific genomic information. The CODEC study is a prospective pilot study to assess the utility of ctDNA as a prognostic biomarker in EC with the hope that it may help predict relapse and guide selection of adjuvant therapy. Methodology: Women with Type 2 EC planned for surgery and consenting to the collection of biospecimens were enrolled prior to surgery and followed prospectively. Blood was collected perioperatively and then every three months for up to two years and at relapse. Broad sequencing of participants’ tumors was performed, the results of which guided the design of personalized droplet digital PCR (ddPCR) assays for ctDNA analysis. ctDNA was extracted from 5ml of plasma and tested in quadruplicate. PET defined metabolic tumor volume was compared to ctDNA and analysis of ctDNA and relapse free survival (RFS) performed using a Cox proportional hazards regression model. Results: Somatic mutations to guide ddPCR analysis were detected in 45 enrolled women stages 1- 4 including pathogenic variants in POLE. ctDNA results are currently available for 30 participants (remaining results awaited). 12/30 participants had detectable ctDNA preoperatively and 5/25 at Time Point 3 (2-6 weeks post-surgery). ctDNA levels varied significantly across the cohort but results to date indicate that perioperative ctDNA positivity is associated with relapse. Interestingly in those that had detectable ctDNA preoperatively, the majority had a rise in ctDNA intraoperatively. Conclusion: This proof-of-concept analysis showed that ctDNA can successfully be detected in the perioperative setting and is the first study in EC to compare ctDNA with PET findings. Although testing is still underway, the early results indicate that ctDNA maybe a useful biomarker to predict relapse and guide the selection of adjuvant therapy. Citation Format: Rachel Delahunty, Rainier Arnolda, Thuan Tzen Koh, Michael Hofman, Hayley Johnston, Patricia Wojtowicz, Caitlyn Nguyen, Nicole Brooks, Kym Reid, Julie Lamont, Thomas Jobling, Deborah Neesham, Vanessa Obers, Clair Shadbolt, Brooke Sawyer, Paul Smith, Jennifer Ellis, Geradline Goss, Mila Volcheck, Madawa Jayawardana, Sandun Silva, Chelsee Hewitt, Kate Jones, Andrew Fellowes, Orla McNally, Stephen Wong, Elizabeth Christie, Costas Yannakou. Assessing Circulating TumOur DNA as a prognostic biomarker in endometrial cancer (The CODEC Study) [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B019.

Development of a duplex droplet digital PCR assay for the detection of Burkholderia cepacia complex and Stenotrophomonas maltophilia in bloodstream infections.

Burkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are nosocomial pathogens that cause various infections and exhibit high resistance to multiple antimicrobial agents. In this study, we aimed to develop a duplex droplet digital PCR (ddPCR) assay for detecting BCC and S. maltophilia in bloodstream infections. We optimized the experimental conditions by setting the annealing temperature to 51°C and determining the optimal concentrations of primers and probes, as well as the thermal cycle numbers. The feasibility of the duplex ddPCR reaction system with the optimal conditions was established and verified through parallel reactions with reference strains of BCC and S. maltophilia. The specificity of the assay, tested with 33 reference strains, was found to be 100%. The duplex ddPCR assay demonstrated good repeatability and could detect as low as 5.35 copies/reaction of BCC and 7.67 copies/reaction of S. maltophilia. This level of sensitivity was consistent in the simulated blood and blood bottle samples. We compared nucleic acid extraction methods and found that the Chelex-100 boiling method and kit extraction method exhibited similar detection sensitivity, suggesting the potential application of the Chelex-100 boiling method in the ddPCR assay. In the clinical samples, the duplex ddPCR assay accurately detected BCC and S. maltophilia in 58 cases. In conclusion, our study successfully developed a duplex ddPCR assay that provides accurate and convenient detection of BCC and S. maltophilia in bloodstream infections.IMPORTANCEBurkholderia cepacia complex (BCC) and Stenotrophomonas maltophilia are implicated in a wide range of infections, including bloodstream infections (BSIs), pneumonia, and meningitis, and often exhibit high intrinsic resistance to multiple antimicrobial agents, limiting therapeutic options. The gold standard for diagnosing bloodstream infections remains blood culture. However, current blood culture detection and positivity rates do not meet the "rapid diagnosis" required for the diagnosis and treatment of critically ill patients with BSIs. The digital droplet PCR (ddPCR) method is a potentially more powerful tool in the diagnosis of BSIs compared to other molecular methods due to its greater sensitivity, specificity, accuracy, and reproducibility. In this study, a duplex ddPCR assay for the detection of BCC and S. maltophilia in BSIs was developed.

E2F1 promotes cell migration in hepatocellular carcinoma via FNDC3B.

FNDC3B (fibronectin type III domain containing 3B) is highly expressed in hepatocellular carcinoma (HCC) and other cancer types, and fusion genes involving FNDC3B have been identified in HCC and leukemia. Growing evidence suggests the significance of FNDC3B in tumorigenesis, particularly in cell migration and tumor metastasis. However, its regulatory mechanisms remain elusive. In this study, we employed bioinformatic, gene regulation, and protein-DNA interaction screening to investigate the transcription factors (TFs) involved in regulating FNDC3B. Initially, 338 candidate TFs were selected based on previous chromatin immunoprecipitation (ChIP)-seq experiments available in public domain databases. Through TF knockdown screening and ChIP coupled with Droplet Digital PCR assays, we identified that E2F1 (E2F transcription factor 1) is crucial for the activation of FNDC3B. Overexpression or knockdown of E2F1 significantly impacts the expression of FNDC3B. In conclusion, our study elucidated the mechanistic link between FNDC3B and E2F1. These findings contribute to a better understanding of FNDC3B in tumorigenesis and provide insights into potential therapeutic targets for cancer treatment.

Combination Assay of Methylated HOXA1 with Tumor Markers Shows High Sensitivity for Detection of Early-Stage Hepatocellular Carcinoma

Introduction: Patients with hepatitis virus-related hepatocellular carcinoma (viral HCC) are decreasing as hepatitis control improves, but those with non-viral-related HCC (non-viral HCC) are increasing in Japan. No established surveillance system exists for patients with non-viral HCC, so they are often diagnosed at an advanced stage. To address this, we performed this study. Methods: We collected serum samples from 516 participants (154 healthy subjects, 93 chronic liver disease [CLD] patients without HCC, and 269 HCC patients). Participants were divided into a control group comprising healthy subjects and patients with CLD and an HCC group. We evaluated serum methylated HOXA1 (m-HOXA1) copy numbers using modified combined restriction digital PCR (CORD) assay (1-step CORD assay). We assessed diagnostic performance of m-HOXA1 compared to HCC tumor markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) and created a novel index to improve HCC prediction. Results: Serum m-HOXA1 level was significantly higher in each HCC stage group versus the control group. Its sensitivity was 69.1% and specificity was 78.5% for diagnosing HCC. The area under the curve (AUC) of m-HOXA1 was superior to that of AFP and equal to that of DCP. Multivariate logistic regression analysis revealed independent contributions of m-HOXA1, DCP, and AFP, in that order of strength, to diagnose HCC after adjustment for age and sex. We designated the predictive probability of HCC based on the regression model as the ASDAm-H1 (Age, Sex, DCP, AFP, and m-HOXA1) index. Its diagnostic accuracy was 0.96 by AUC with a sensitivity of 86.2% and specificity of 93.9%. Sensitivity was identical for viral and non-viral HCCs. When limited to early-stage HCC, sensitivity of the ASDAm-H1 index was 76.3%. Conclusions: We showed distinguished performance of the ASDAm-H1 index to detect viral and non-viral HCC, even at an early stage. This index might have potential as a non-viral HCC surveillance system.

Improved Canker Processing and Viability Droplet Digital PCR Allow Detection of Erwinia amylovora Viable Nonculturable Cells in Apple Bark.

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 102 to 7.6 × 107 cells mL-1, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management.

CtDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.

Clinically accessible amplitude-based multiplex ddPCR assay for tryptase genotyping

Hereditary α tryptasemia (HαT) is an autosomal dominant trait characterized by increased TPSAB1 copy number (CN) encoding α-tryptase. The determination of HαT is being discussed as an important biomarker to be included in risk assessment models and future diagnostic algorithms for patients with mastocytosis and anaphylaxis. Due to the complex genetic structure at the human tryptase locus, genetic testing for tryptase gene composition is presently notably limited and infrequently pursued. This study aimed to develop, optimise and validate a multiplex droplet digital PCR (ddPCR) assay that can reliably quantify α- and β-tryptase encoding sequences in a single reaction. To optimise the ddPCR conditions and establish an amplitude-based multiplex ddPCR assay, additional primers and probes, a thermal gradient with varying annealing temperatures, different primers/probe concentrations, and various initial DNA quantities were tested. Results obtained from all 114 samples analysed using multiplex ddPCR were identical to those obtained through the use of original duplex assays. Utilizing this multiplex ddPCR assay, in contrast to conducting distinct duplex ddPCRs, presents noteworthy benefits for tryptase genotyping. These advantages encompass a substantial threefold decrease in material costs and considerable time savings. Consequently, this approach exhibits high suitability and particularly captures interest for routine clinical implementation.

Plasma-based analysis of ERBB2 mutational status by multiplex digital PCR in a large series of patients with metastatic breast cancer.

Erb-b2 receptor tyrosine kinase 2 (ERBB2)-activating mutations are therapeutically actionable alterations found in various cancers, including metastatic breast cancer (MBC). We developed multiplex digital PCR assays to detect and quantify ERBB2 mutations in circulating tumor DNA from liquid biopsies. We studied the plasma from 272 patients with hormone-receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) MBC to detect 17 ERBB2 mutations using a screening assay. The assay was developed on the three-color Crystal dPCR™ naica® platform with a two-step strategy for precise mutation identification. We found that nine patients (3.3%) harbored at least one ERBB2 mutation. The mutation rate was higher in patients with lobular histology (5.9%) compared to invasive breast carcinoma of no special type (2.6%). A total of 12 mutations were found with the following frequencies: L755S (25.00%), V777L (25.00%), S310Y (16.67%), L869R (16.67%), S310F (8.33%), and D769H (8.33%). Matched tumor samples from six patients identified the same mutations with an 83% concordance rate. In summary, our highly sensitive multiplex digital PCR assays are well suited for plasma-based monitoring of ERBB2 mutational status in patients with MBC.

Development and validation of a novel multiplex digital PCR assay for identification of pathogens in cerebrospinal fluid of children with bacterial meningitis

Background and aimsIdentifying the pathogens of bacterial meningitis (BM) is crucial for its diagnosis and treatment. The aim of this study is to develop and validate a novel method for detecting pathogens in cerebrospinal fluid (CSF) of children with BM using a digital polymerase chain reaction (dPCR) assay.Materials and methods:A novel multiplex dPCR assay method has been developed and validated. The diagnostic performance of the dPCR assay was compared with that of synchronous CSF culture, and the factors affecting its performance were analyzed.Results:A total of 69 children with BM were enrolled prospectively. The sensitivity of the dPCR assay was 94.44 %, specificity was 100 %, coincidence rate was 98.55 %, Kappa value was 0.959, and net reclassification improvement was 61.11 %. Compared with the CSF culture assay, the dPCR assay had higher sensitivity in different bacterial groups. Multiple factors affected its performance, including previous use of antibiotics, sampling time, BM complications, and levels of inflammatory biomarkers in CSF and blood (all P < 0.05). Patients who required intensive care and died had a higher bacterial DNA loads identified by dPCR assay (both P < 0.05).Conclusion:This novel assay has better pathogen detection ability than CSF culture. Its performance was influenced by sampling time, previous use of antibiotics, and disease severity.

An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay

To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

Digital PCR Improves Sensitivity and Quantification in Monitoring CAR-T Cells in B Cell Lymphoma Patients

Chimeric antigen receptor T-cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analysed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC.

Digital PCR assay for the specific detection and estimation of Salmonella contamination levels in poultry rinse

Strains of Salmonella are a frequent cause of foodborne illness and are known to contaminate poultry products. Most Salmonella testing methods can qualitatively detect Salmonella and cannot quantify or estimate the Salmonella load in samples. Therefore, the aim of this study was to standardize and validate a partitioned-based digital PCR (dPCR) assay for the detection and estimation of Salmonella contamination levels in poultry rinses. Pure culture Salmonella strains were cultured, enumerated, cold-stressed for 48 h, and used to inoculate whole carcass chicken rinse (WCCR) at 1–4 log CFU/30 mL and enriched at 37 °C for 5 h. Undiluted DNA samples with primer and probes targeting the Salmonella-specific invA gene were used for the dPCR assay. The dPCR assay was highly specific, with a limit of detection of 0.001 ng/μL and a limit of quantification of 0.01 ng/μL. The dPCR assay further showed no PCR reaction inhibition up to 5 μg of crude DNA extract. The assays accurately detected all cold-stressed Salmonella in inoculated WCCR samples following a 5-h enrichment. Most importantly, when converted to log, the dPCR copies/μL values accurately estimated the inoculated Salmonella levels. The dPCR assay standardized in this study is a robust method for the detection and estimation of Salmonella concentration in contaminated food samples. This approach can allow same-day decision-making for poultry processors attempting to maintain limits and controls on Salmonella contamination.

Analytical and clinical validation of multiplex droplet digital PCR assay for detecting pathogenic fungal infection in lungs

ABSTRACT Pulmonary invasive fungal infection in immunocompromised hosts is difficult to diagnose, and current tools for diagnosis or monitoring of response to antifungal treatments have inherent limitations. Droplet digital PCR (ddPCR) has emerged as a promising tool for pulmonary pathogen detection with high sensitivity. This study presents a novel ddPCR panel for rapid and sensitive identification of pulmonary fungal pathogens. First, a ddPCR method for detecting three fungal genera, including Pneumocystis, Aspergillus, and Cryptococcus, was established and evaluated. Then, the clinical validation performance of ddPCR was compared with that of qPCR using 170 specimens, and the 6 specimens with inconsistent results were further verified by metagenomics next-generation sequencing, which yielded results consistent with the ddPCR findings. Finally, the area under the ROC curve (AUC) was used to evaluate the efficiency of ddPCR. While the qPCR identified 16 (9.41%) cases of Aspergillus and 6 (3.53%) cases of Pneumocystis, ddPCR detected 20 (11.76%) Aspergillus cases and 8 (4.71%) Pneumocystis cases. The AUC for Aspergillus, Cryptococcus, and Pneumocystis was 0.974, 0.998, and 0.975, respectively. These findings demonstrated that the ddPCR assay is a highly sensitive method for identifying pathogens responsible for invasive fungal pulmonary infections, and is a promising tool for early diagnosis.