UNASSIGNED: CYP2D6 testing is increasingly used to guide drug therapy and thus, reliable methods are needed to test this complex and polymorphic gene locus. A particular challenge arises from the detection and interpretation of structural variants (SVs) including gene deletions, duplications, and hybrids with the CYP2D7 pseudogene. This study validated the Absolute Q™ platform for digital PCR-based CYP2D6 copy number variation (CNV) determination by comparing results to those obtained with a previously established method using the QX200 platform. In addition, protocols for streamlining CYP2D6 CNV testing were established and validated including the \"One-pot\" single-step restriction enzyme digestion and a multiplex assay simultaneously targeting the CYP2D6 5\'UTR, intron 6, and exon 9 regions.
UNASSIGNED: Genomic DNA (gDNA) samples from Coriell (n = 13) and from blood, saliva, and liver tissue (n = 17) representing 0-6 copies were tested on the Absolute Q and QX200 platforms. Custom TaqMan™ copy number (CN) assays targeting CYP2D6 the 5\'UTR, intron 6, and exon 9 regions and a reference gene assay (TERT or RNaseP) were combined for multiplexing by optical channel. In addition, two digestion methods (One-pot digestion and traditional) were assessed. Inconclusive CN values on the Absolute Q were resolved using an alternate reference gene and/or diluting gDNA.
UNASSIGNED: Overall, results between the two platforms and digestions methods were consistent. The \"One-pot\" digestion method and optically multiplexing up to three CYP2D6 regions yielded consistent result across DNA sample types and diverse SVs, reliably detecting up to 6 gene copies. Rare variation in reference genes were found to interfere with results and interpretation, which were resolved by using a different reference.
UNASSIGNED: The Absolute Q produced accurate and reliable CYP2D6 copy number results allowing for a streamlined and economical protocol using One-pot digestion and multiplexing three target regions. Protocols are currently being expanded to other pharmacogenes presenting with SVs/CNVs.

Circulating tumor DNA (ctDNA) is naked DNA molecules shed from the tumor cells into the peripheral blood circulation. They contain tumor-specific gene mutations and other valuable information. ctDNA is considered to be one of the most significant analytes in liquid biopsies. Over the past decades, numerous researchers have developed various detection strategies to perform quantitative or qualitative ctDNA analysis, including PCR-based detection and sequencing-based detection. More and more studies have illustrated the great value of ctDNA as a biomarker in the diagnosis, prognosis and heterogeneity of tumor. In this review, we first outlined the development of digital PCR (dPCR)-based and next generation sequencing (NGS)-based ctDNA detection systems. Besides, we presented the introduction of the emerging ctDNA analysis strategies based on various biosensors, such as electrochemical biosensors, fluorescent biosensors, surface plasmon resonance and Raman spectroscopy, as well as their applications in the field of biomedicine. Finally, we summarized the essentials of the preceding discussions, and the existing challenges and prospects for the future are also involved.

BACKGROUND: Accurate estimation of cell viability is crucial in various applications such as cytotoxicity testing and routine cell culture on both industrial and laboratory scales. For this, the real-time monitoring of cell status would be beneficial. Conventional cell-based assays for cell viability have limitations in sensitivity and time-effectiveness. Analysis of cell-free DNA (cfDNA) in (culture) media is a good alternative as cfDNA release are a well-known phenomenon during cell death.
RESULTS: We demonstrate a direct digital PCR (dPCR) method to estimate cell viability by analyzing cfDNA in media during induced cell death. After validating the duplex dPCR method for short and long amplicons of the SMAD4 and RPP30 loci, we determined that a media volume of 2 μL is feasible to measure the target DNA copy number with minimal negative effects on amplification. dPCR inhibition was evident with a higher media volume per reaction targeting long amplicons. Next, we applied our dPCR method using media cfDNA and other conventional methods to the monitoring of camptothecin (CPT)-induced cell death. Copy numbers increased significantly after 4 h of CPT treatment, showing a fold change of approximately 4-6 compared to the controls. Cell-based assays such as the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and annexin V/7-AAD assay also indicated increased cell death at 4 h, but the trypan blue exclusion assay did not.
CONCLUSIONS: The developed media cfDNA direct dPCR method allows for efficient measurements of the degree of cell viability. Unlike other conventional cell-based assays, our method has advantages of no loss of cultured cells and the ability to implement online analysis. Accurate and sensitive media cfDNA analysis using dPCR can be adopted in various applications such as determining cytotoxicity levels in large-scale bioreactors or screening for effective anticancer drugs.

The management of hematologic malignancies has entered a new era in which minimal residual disease (MRD) monitoring plays a pivotal role. Well-established molecular targets, such as PML::RARA, CBFB::MYH11, or RUNX1::RUNX1T1, are conventionally tracked by quantitative RT-PCR. Recently, a broader landscape of fusion transcripts has been unveiled through transcriptomic analysis. These newly discovered fusion transcripts may emerge as novel molecular markers for MRD quantification. In this study, we compared a targeted RNA-sequencing (RNA-seq) approach (FusionPlex) with a whole-transcriptomic strategy (Advanta RNA-Seq XT) for fusion detection in a training set of 21 samples. We evidenced a concordance of 100% for the detection of known fusions, and showed a good correlation for gene expression quantification between the two techniques (Spearman r = 0.77). Additionally, we prospectively evaluated the identification of fusions by targeted RNA-seq in a real-life series of 126 patients with hematological malignancy. At least one fusion transcript was detected for 60 patients (48%). We designed tailored digital PCR assays for 11 rare fusions, and validated this technique for MRD quantification with a limit of detection of <0.01%. The combination of RNA-seq and tailored digital PCR may become a new standard for MRD evaluation in patients lacking conventional molecular targets.

BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified.
METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages.
RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages.
CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.

Digital polymerase chain reaction (dPCR) has emerged as a groundbreaking technology in molecular biology and diagnostics, offering exceptional precision and sensitivity in nucleic acid detection and quantification. This review highlights the core principles and transformative potential of dPCR, particularly in infectious disease diagnostics and environmental surveillance. Emphasizing its evolution from traditional PCR, dPCR provides accurate absolute quantification of target nucleic acids through advanced partitioning techniques. The review addresses the significant impact of dPCR in sepsis diagnosis and management, showcasing its superior sensitivity and specificity in early pathogen detection and identification of drug-resistant genes. Despite its advantages, challenges such as optimization of experimental conditions, standardization of data analysis workflows, and high costs are discussed. Furthermore, we compare various commercially available dPCR platforms, detailing their features and applications in clinical and research settings. Additionally, the review explores dPCR\'s role in water microbiology, particularly in wastewater surveillance and monitoring of waterborne pathogens, underscoring its importance in public health protection. In conclusion, future prospects of dPCR, including methodological optimization, integration with innovative technologies, and expansion into new sectors like metagenomics, are explored.

Reverse transcription-digital PCR (RT-dPCR) is attracting attention as a method that enables SI-traceable RNA quantification without calibration, but its accuracy and bias have not been thoroughly studied. In this study, the accurate quantification of RNA by the RT-dPCR method was investigated using NMIJ CRM 6204-b, an RNA certified reference material whose certified value was assigned by orthogonal chemical measurement methods. Moreover, a two-step RT-dPCR method was adopted to examine in detail the conditions for the RT reaction process, which was expected to be the major uncertainty component in the RT-dPCR measurement. Optimization experiments revealed that the type of reverse transcriptase, the concentration of template RNA, and the type and concentration of primers in the RT reaction affected the value quantified by RT-dPCR. Under the optimal conditions, the value quantified by RT-dPCR, 76.4 ng/μL ± 6.7 ng/μL (the quantified value ± expanded uncertainty (k = 2)), was consistent with the certified value, 68.2 ng/μL ± 5.8 ng/μL, of NMIJ CRM 6204-b RNA 1000-A within the expanded uncertainty. From the results of the uncertainty evaluation, the relative combined uncertainty of the RT-dPCR method was 4.42%, and the major uncertainty components in the RT-dPCR method were the preparation of RT solution (3.68%), the inter-day difference (1.80%), and the RT reaction (1.30%). Together, the results suggested that the contribution of the RT reaction process to the total uncertainty was greater than that of the dPCR process.

UNASSIGNED: Li-Fraumeni syndrome is a hereditary tumor syndrome characterized by an elevated risk of malignancy, particularly acute lymphoblastic leukemia (ALL), which can be caused by the heterozygous germline mutation. TP53 gene germline mutation is considered a potential risk factor and crucial prognostic parameter for acute leukemia development and diagnosis, but rarely occurs in adults, and its specific pathogenic significance in acute leukemia is unclear.
UNASSIGNED: We describes a case of a 45-year-old woman diagnosed with ALL. Whole-exome sequencing approach identified one of the TP53 germline mutations from her bone marrow sample with possible pathogenic significance, c.848G>A (p.Arg283His) heterozygous missense mutation located on exon 8, which was further verified in her hair, oral mucous and nail samples. Family pedigree screening revealed that the same TP53 genetic variant was present in the patient\'s father and non-donor son, whereas not in the donor. Digital PCR observed that this point mutation frequency dropped post-transplantation but remained low during maintenance therapy when the patient was leukemia-free.
UNASSIGNED: This suspected Li-Fraumeni syndrome case report with a likely pathogenic heterozygous TP53 variant expands the cancer genetic spectrum. Screening her family members for mutations facilitates identifying the optimal relative donor and avoids unnecessary treatment by monitoring TP53 germline mutations for minimal residual disease following hematopoietic stem cell transplantation. Its potential roles in hematological malignant tumor development and clinical pathogenic implications necessitate further probing.

Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir-a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1-MOCV3), and PG qualification (PG1-6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization.