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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. [ABSTRACT FROM AUTHOR]

This article presents a study that compares different techniques for estimating gene copy numbers in genetically modified crops. The researchers evaluated Southern blotting, qPCR, dPCR, and PE-WGS and found that all four methods were suitable to varying degrees for determining gene copy numbers. The strengths and limitations of each technique are discussed, and the authors suggest prioritizing dPCR and PE-WGS for precise analysis. The document also provides references and supporting information for three related articles, which can be helpful for library patrons researching agricultural biotechnology, PCR techniques, and structural variation studies in plants. [Extracted from the article]

Background: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This study utilized digital PCR to quantify HBV RNA and HBV DNA within three regions of the HBV genome. Analysis of 75 serum samples from patients with chronic infection showed that HBV RNA levels were higher in core than in S and X regions (median 7.20 vs. 6.80 and 6.58 log copies/mL; p <.0001), whereas HBV DNA levels showed an inverse gradient (7.71 vs. 7.73 and 7.77 log copies/mL, p <.001). On average 80% of the nucleic acid was DNA by quantification in core. The core DNA/RNA ratio was associated with viral load and genotype. In individual patients, the relations between RNA levels in core, S and X were stable over time (n = 29; p =.006). The results suggest that pregenomic RNA is completely reverse transcribed to minus DNA in ≈75% of the virus particles, whereas the remaining 25% contain both RNA and DNA of lengths that reflect variable progress of the polymerase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. Objective: A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. Methods: In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. Results: Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. Conclusion: The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation. [ABSTRACT FROM AUTHOR]

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Terminalia bellirica (TB), Terminalia chebula (TC), and Phyllanthus emblica (PE) fruits are renowned for their diverse therapeutic benefits, propelling their cultivation and use in herbal remedies. However, the global surge in demand driven by the awareness and long-term benefits of using herbal medicines has inadvertently led to a rise in adulteration practices within the herbal market. Recent advancement in DNA authentication of herbal products is constrained by poor quality and quantity of PCR amplifiable DNA obtained from the dried and polyphenol-rich fruits of processed herbal products, resulting in inconsistent PCR amplification due to heterogeneous secondary metabolites. This study tailored a DNA isolation protocol by optimizing buffering strength to stabilize pH and adding phenolic compound scavenger additives, such as polyvinylpyrrolidone, during the cell lysis step. The implemented procedure resulted in significant enhancements in both the quantity and quality of PCR amplifiable DNA. PCR amenability was evaluated using ITS2 metabarcode. Later, species-specific assays, targeting ITS-based SCAR markers specific to TB, TC, and PE, were performed on six market powders for each plant species. TB, TC, and PE were detected in 100, 83.3, and 50% of the six market samples, respectively. Digital PCR increases the assay's sensitivity by two-fold compared to conventional PCR. To the best of our knowledge, this is the first instance of utilizing dPCR for authenticating TB, TC, and PE fruits. The improvised DNA extraction protocol successfully demonstrates how a comprehensive analysis of PCR amplifiable DNA isolation and PCR dynamics enables the effective resolution of challenges related to poor DNA quality and quantity, as well as the inconsistency encountered during PCR due to the heterogeneity of polyphenols. [ABSTRACT FROM AUTHOR]

BackgroundThe relationship between the viral kinetics of SARS-CoV-2 and clinical outcomes remains unclear.MethodsA convenience sample of 955 remnant nasopharyngeal swabs collected during routine care between 11/18/20 and 9/26/21 were analyzed using digital PCR and associated clinical data extracted from the medical record. 18 individuals had >1 sample within 30 days of onset of symptoms.ResultsPaired samples were an average of 6 [range: 0-13] days apart. Four individuals sampled twice on the same day had a median 0.52 log10 viral load difference between samples. Of the remaining, 12 individuals had a decrease in viral load over time, with an average decay of -0.23 log10/day.ConclusionsOur study found a similar rate of viral decay to others, but did not find associations between viral kinetics and clinical outcomes. Larger studies would be useful to support the use of this measurement as a surrogate endpoint for therapeutic studies.

Abstract 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. Graphical Abstract

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (HIV reservoir). One such technique for HIV RNA quantitation, HIV transcription profiling, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the HIV transcription profiling technique to Qiagens dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

Using genetically modified (GM) soybean MON87705 containing the figwort mosaic virus 35S promoter (FMV 35S) as a marker gene, a quantitative method for detecting transgenic crops was established based on real-time fluorescent quantitative polymerase chain reaction (real-time PCR) and digital polymerase chain reaction (dPCR). The amplification system was tested by real-time PCR, revealing that the developed method was highly specific to FMV 35S. By determining quantitative parameters using duplex droplet digital PCR (ddPCR), it was found that within the genomic DNA concentration range of 0.005–20 ng/mL, the measured copy number of the target gene had a good linear correlation with the theoretical copy number; the limit of detection (LOD) and the limit of quantification (LOQ) of dPCR for FMV 35S were as low as 5 copies and 0.1%, respectively. Blind samples with different contents of MON87705 were tested on two dPCR platforms, indicating that accurate, reliable and reproducible results were obtained in the duplex and triple detection using a combination of internal reference genes and foreign specific fragments. Therefore, the quantitative dPCR method, which can meet the parameter requirements of the quantitative detection standard for genetically modified components with improved detection efficiency, represents an accurate and simple technology for the quantitative detection of genetically modified crops.

Pleural mesothelioma (PM) is a highly aggressive tumor that is caused by asbestos exposure and lacks effective therapeutic regimens. Current procedures for PM diagnosis are invasive and can take a long time to reach a definitive result. Small extracellular vesicles (sEVs) have been identified as important communicators between tumor cells and their microenvironment via their cargo including circular RNAs (circRNAs). CircRNAs are thermodynamically stable, highly conserved, and have been found to be dysregulated in cancer. This study aimed to identify potential biomarkers for PM diagnosis by investigating the expression of specific circRNA gene pattern (hsa_circ_0007386) in cells and sEVs using digital polymerase chain reaction (dPCR). For this reason, 5 PM, 14 non-PM, and one normal mesothelial cell line were cultured. The sEV was isolated from the cells using the gold standard ultracentrifuge method. The RNA was extracted from both cells and sEVs, cDNA was synthesized, and dPCR was run. Results showed that hsa_circ_0007386 was significantly overexpressed in PM cell lines and sEVs compared to non-PM and normal mesothelial cell lines (p < 0.0001). The upregulation of hsa_circ_0007386 in PM highlights its potential as a diagnostic biomarker. This study underscores the importance and potential of circRNAs and sEVs as cancer diagnostic tools. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all‐in‐one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation‐induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette‐based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning‐driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high‐multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost‐effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew- |$t$| distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app. [ABSTRACT FROM AUTHOR]

Infectious diseases account for about 3 million deaths per year. The advent of molecular techniques has led to an enormous improvement in their diagnosis, both in terms of sensitivity and specificity and in terms of the speed with which a clinically useful result can be obtained. Digital PCR, or 3rd generation PCR, is based on a series of technical modifications that result in more sensitive techniques, more resistant to the action of inhibitors and capable of direct quantification without the need for standard curves. This review presents the main applications that have been developed for the diagnosis of viral, bacterial, and parasitic infections and the potential prospects for the clinical use of this technology. [ABSTRACT FROM AUTHOR]

Sweet potato leaf curl virus (SPLCV) is a whitefly-transmitted begomovirus infecting sweetpotato and other morning glory (Convolvulaceae) species worldwide. The virus is widespread at the USDA, ARS, U.S. Vegetable Laboratory (USVL), and testing of germplasm maintained in the breeding program indicates nearly 100% infection in storage roots of materials propagated for at least four years. Prior to the public release of new germplasm, viruses must be eliminated via laborious and time-consuming meristem-tip culture. The identification of virus-free seedlings early in the selection process can offer an alternative to meristem-tip culture. In this study, we investigated the transmission of SPLCV over two years of consecutive field plantings (early and late) of sweetpotato. While SPLCV is endemic at the USVL, virus transmission pressure over the typical cultivation season is unknown, and avoidance of virus transmission paired with the selection and maintenance of clean material may be a viable alternative to virus elimination. In 2022, the storage roots of 39 first-year seedling (FYS) selections were tested for SPLCV after early-season cultivation, revealing a single selection (2.6%) with a positive test. Similar testing was conducted in 2023 with no SPLCV-positive FYS selections detected. To further assess SPLCV acquisition in the field, replicated late-season plantings of each selected FYS (n = 37) were monitored from planting to harvest. Testing was conducted at 60 and 120 days after planting (DAP). Approximately 35% of the bulk samples were infected at 60 DAP, and infection increased to 52.3% by 120 DAP. Testing of individuals within selected positive bulked samples did not support 100% infection at harvest. Altogether, these results demonstrate that SPLCV transmission during early planting is sufficiently low to facilitate the maintenance of virus-free selections, offering an alternative to virus cleaning and a cultivation strategy that may be leveraged for production. [ABSTRACT FROM AUTHOR]

This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.Key Message: Optimized transformation protocol led to high hairy root formation and T-DNA-gene integration rates. Ri plants were successfully regenerated for ‘M26’ and ‘Gala’, differing in integrated T-DNA genes and copy numbers. [ABSTRACT FROM AUTHOR]

The unprecedented demand for variants diagnosis in response to the COVID‐19 epidemic has brought the spotlight onto rapid and accurate detection assays for single nucleotide polymorphisms (SNPs) at multiple locations. However, it is still challenging to ensure simplicity, affordability, and compatibility with multiplexing. Here, a novel technique is presented that combines peptide nucleic acid (PNA) clamps and near‐infrared (NIR)‐driven digital polymerase chain reaction (dPCR) to identify the Omicron and Delta variants. This is achieved by simultaneously identifying highly conserved mutated signatures at codons 19, 614, and 655 of the spike protein gene. By microfluidically introducing graphene‐oxide‐nanocomposite into the assembled gelatin microcarriers, they achieved a rapid temperature ramping‐up rate and switchable gel‐to‐sol phase transformation synchronized with PCR activation under NIR irradiation. Two sets of duplex PCR reactions, each classifying respective PNA probes, are emulsified in parallel and illuminated together using a homemade vacuum‐based droplet generation device and a programmable NIR control module. This allowed for selective amplification of mutant sequences due to single‐base‐pair mismatch with PNA blockers. Sequence‐recognized bioreactions and fluorescent‐color scoring enabled quick identification of variants. This technique achieved a detection limit of 5,100 copies and a 5‐fold quantitative resolution, which is promising to unfold minor differences and dynamic changes. [ABSTRACT FROM AUTHOR]

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life. [ABSTRACT FROM AUTHOR]

Simple Summary: A multiplexed optimised bias-based preamplification–digital droplet PCR (OBBPA-ddPCR) diagnostic technique was developed and tested in this study. The method estimates blood-derived cell-free DNA (cfDNA) methylation levels of RASSF1A, MIR129-2, NRIP3, and SOX8 target sequences in blood samples. The study included 90 healthy individuals, 40 benign prostatic hyperplasia patients, and 39 prostate cancer (PCa) patients. The risk scores were developed to further reduce unnecessary prostate biopsies and PCa overdiagnosis. The scores included classical risk factors and methylation data, delivering an improved 70% specificity for BPH patients and 100% sensitivity for clinically significant PCa patients. Current prostate carcinoma (PCa) biomarkers, including total prostate-specific antigen (tPSA), have unsatisfactory diagnostic sensitivity and specificity resulting in overdiagnosis and overtreatment. Previously, we described an optimised bias-based preamplification–digital droplet PCR (OBBPA-ddPCR) technique, which detects tumour DNA in blood-derived cell-free DNA (cfDNA) of cancer patients. The current study investigated the performance of newly developed OBBPA-ddPCR-based biomarkers. Blood plasma samples from healthy individuals (n = 90, controls) and PCa (n = 39) and benign prostatic hyperplasia patients (BPH, n = 40) were analysed. PCa and BPH patients had tPSA values within a diagnostic grey area of 2–15 ng/mL, for whom further diagnostic validation is most crucial. Methylation levels of biomarkers RASSF1A, MIR129-2, NRIP3, and SOX8 were found significantly increased in PCa patients compared to controls. By combining classical PCa risk factors (percentage of free PSA compared to tPSA (QfPSA) and patient's age) with cfDNA-based biomarkers, we developed PCa risk scores with improved sensitivity and specificity compared to established tPSA and QfPSA single-marker analyses. The diagnostic specificity was increased to 70% with 100% sensitivity for clinically significant PCa patients. Thus, prostate biopsies could be avoided for 28 out of 40 BPH patients. In conclusion, the newly developed risk scores may help to confirm the clinical decision and prevent unnecessary prostate biopsy. [ABSTRACT FROM AUTHOR]

Currently, there are no reliable prognostic factors to determine which upper tract urothelial carcinoma (UTUC) patients will progress after radical nephroureterectomy (RNU). We aim to evaluate whether liquid-biopsy-based biomarkers (circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulating tumor DNA (ctDNA)) were able to predict clinical outcomes in localized UTUC patients undergoing RNU. Twenty patients were prospectively enrolled between 2021 and 2023. Two blood samples were collected before RNU and three months later. CTCs and cfDNA were isolated and evaluated using the IsoFlux system and Quant-iT PicoGreen dsDNA kit, respectively. Droplet digital PCR was performed to determine ctDNA status. Cox regression analysis was performed on CTCs, cfDNA, and ctDNA at two different follow-up time points to examine their influence on tumor progression and cancer-specific survival (CSS). During a median follow-up of 18 months, seven (35%) patients progressed and three (15%) died. Multivariate analysis demonstrated that cfDNA levels three months after RNU are a significant predictor of tumor progression (HR = 1.085; p = 0.006) and CSS (HR = 1.168; p = 0.029). No associations were found between CTC enumeration and ctDNA status with any of the clinical outcomes evaluated. The evaluation of cfDNA levels in clinical practice could improve the disease management of UTUC patients. [ABSTRACT FROM AUTHOR]

The rapid and sensitive detection of foodborne pathogens is crucial for ensuring food safety. Among virus testing methods, polymerase chain reaction (PCR) has served as the gold-standard technique in most food safety regulation organizations. However, to enhance the speed and efficiency of PCR, novel approaches are continually being explored. In this work, leveraging the photothermal effects and high thermal conductivity of gold nanoparticles, we have significantly improved the heating and cooling rates of thermal cycles, enabling ultra-fast PCR detection. Specifically, we present a pre-degassing multiplex digital PCR chip integrated with gold nanoparticles. We further developed a portable system with a light source for photothermal heating cycling, along with an optoelectronic sensor to analyze PCR amplification products after rapid thermal cycling. As proof of concept, the proposed chip and portable device was applied for the on-site detection of several types of foodborne pathogens, including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, and Salmonella. The whole system could distinguish those pathogens within 20 min, showing good potential for the rapid detection of multiple types of foodborne pathogens. [ABSTRACT FROM AUTHOR]

In vivo expansion of genetically modified T cells in cancer patients following adoptive transfer has been linked to both anti-tumor activity and T cell-mediated toxicities. The development of digital PCR has improved the accuracy in quantifying the in vivo status of adoptively infused T cells compared to qPCR or flow cytometry. Here, we developed and evaluated the feasibility and performance of nanoplate-based digital PCR (ndPCR) to quantify adoptively infused T cells engineered with a T cell receptor (TCR) that recognizes a human endogenous retrovirus type E (HERV-E) antigen. Analysis of blood samples collected from patients with metastatic kidney cancer following the infusion of HERV-E TCR-transduced T cells established the limit of detection of ndPCR to be 0.3 transgene copies/μL of reaction. The lower limit of quantification for ndPCR was one engineered T cell per 10,000 PBMCs, which outperformed both qPCR and flow cytometry by 1 log. High inter-test and test-retest reliability was confirmed by analyzing blood samples collected from multiple patients. In conclusion, we demonstrated the feasibility of ndPCR for detecting and monitoring the fate of TCR-engineered T cells in adoptive cell therapy.

BackgroundCYP2D6 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.MethodsGenomic 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.ResultsOverall, 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.ConclusionThe 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.

ABSTRACT The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2–9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6–17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with

The international working group "Development of methods for identification of foodstuffs produced by means of genetic engineering techniques" was established pursuant to § 64 of the German Food and Feed Code (LFGB). It has developed a guideline for implementing and verifying digital PCR methods previously validated as real-time PCR techniques. While event-specific methods in testing of genetically modified organisms (GMO) have traditionally relied on real-time PCR, new digital PCR technologies offer significant advantages and therefore, leading to an increased use by control laboratories. This guideline provides practical recommendations for transferring the real-time PCR to digital PCR and for verifying the digital PCR method. The guideline is applicable to analysis of GMO in food, feed and seed and is freely available on the website of the Federal Office of Consumer Protection and Food Safety (BVL). [ABSTRACT FROM AUTHOR]

Abstract The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction (PCR) assays on patients’ respiratory specimens, which are collected through either nasal or oropharyngeal swabs. The manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper sampling. In this paper, we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab sampling. The soft end-effector utilizes circumferential instability to ensure grasping stability, and exhibits several key properties, including high load-to-weight ratio, error tolerance, and variable swab-tip stiffness, leading to successful automatic robotic oropharyngeal swab sampling, from loosening and tightening the transport medium tube cap, holding the swab, and conducting sampling, to snapping off the swab tail and sterilizing itself. Using an industrial collaborative robotic arm, we integrated the soft end-effector, force sensor, camera, lights, and remote-control stick, and developed a robotic oropharyngeal swab sampling system. Using this swab sampling system, we conducted oropharyngeal swab-sampling tests on 20 volunteers. Our Digital PCR assay results (RNase P RNA gene absolute copy numbers for the samples) revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease diagnosis. In summary, we have developed a pharyngeal swab-sampling system based on an “enveloping” soft actuator, studied the sampling process, and implemented whole-process robotic oropharyngeal swab-sampling.

Abstract Background Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. Methods ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics. Results The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125–225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105–7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. Conclusions dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2

Abstract Background The 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. Methods To 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. Results In 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. Conclusions Our 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.

Backgroud 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.Case presentation 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.Conclusion 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.

The most widely adopted method for diagnosing respiratory infectious diseases is to conduct polymerase chain reaction (PCR) assays on patients' respiratory specimens, which are collected through either nasal or oropharyngeal swabs. The manual swab sampling process poses a high risk to the examiner and may cause false-negative results owing to improper sampling. In this paper, we propose a pneumatically actuated soft end-effector specifically designed to achieve all of the tasks involved in swab sampling. The soft end-effector utilizes circumferential instability to ensure grasping stability, and exhibits several key properties, including high load-to-weight ratio, error tolerance, and variable swab-tip stiffness, leading to successful automatic robotic oropharyngeal swab sampling, from loosening and tightening the transport medium tube cap, holding the swab, and conducting sampling, to snapping off the swab tail and sterilizing itself. Using an industrial collaborative robotic arm, we integrated the soft end-effector, force sensor, camera, lights, and remote-control stick, and developed a robotic oropharyngeal swab sampling system. Using this swab sampling system, we conducted oropharyngeal swab-sampling tests on 20 volunteers. Our Digital PCR assay results (RNase P RNA gene absolute copy numbers for the samples) revealed that our system successfully collected sufficient numbers of cells from the pharyngeal wall for respiratory disease diagnosis. In summary, we have developed a pharyngeal swab-sampling system based on an "enveloping" soft actuator, studied the sampling process, and implemented whole-process robotic oropharyngeal swab-sampling. [ABSTRACT FROM AUTHOR]

Background: Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. Methods: ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics. Results: The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125–225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105–7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. Conclusions: dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments. [ABSTRACT FROM AUTHOR]

Introduction: Induced modification of plant gene expression is of both fundamental and applied importance. Cis-acting regulatory elements (CREs) are major determinants of the spatiotemporal strength of gene expression. Yet, there are few examples where induced genetic variation in predetermined CREs has been exploited to improve or investigate crop plants. Methods: The digital PCR based FIND-IT technology was applied to discover barley mutants with CRE variants in the promoter of the nutritional important barley grain phytase (PAPhy_a) gene. Results and discussion: Mutants with higher or lower gene expression and ultimately higher or lower mature grain phytase activity (MGPA), respectively, were discovered. Field trials and inositol phosphate profiling during germination showed that PAPhy_a does not influence agronomic performance under the trial conditions but it does shorten the lag time of phosphate mobilization during germination. Higher endogenous MGPA is an improvement of grain quality for feed use as it improves the phosphate bioavailability for monogastric animals. Moreover, as the targeted CRE motifs of the PAPhy_a promoter are shared with a range of seed expressed genes like key cereal and legume storage genes, the current results demonstrates a concept for modulating individual gene expression levels of a range of seed genes. [ABSTRACT FROM AUTHOR]

Background: The 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. Methods: To 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. Results: In 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. Conclusions: Our 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioma is one of the most common primary central nervous system (CNS) tumors, and its molecular diagnosis is crucial. However, surgical resection or biopsy is risky when the tumor is located deep in the brain or brainstem. Liquid biopsy, particularly targeting cell-free DNA (cfDNA) in cerebrospinal fluid (CSF), is a minimally invasive method, and emerges as a promising alternative, overcoming spatial and temporal heterogeneity in CNS tumors. Unlike extracranial cancers, cfDNA in the blood is scarce in CNS tumors, including glioma, emphasizing the relevance of CSF. Several successful studies have been conducted to detect tumor-specific genetic alterations in cfDNA from CSF using digital PCR and/or next-generation sequencing. This review provides an overview of the current status of CSF-based cfDNA-targeted liquid biopsy for gliomas. It delineates distinctions from liquid biopsies for extracranial cancers, addresses prevailing challenges, and explores future prospects in this field. Glioma is one of the most common primary central nervous system (CNS) tumors, and its molecular diagnosis is crucial. However, surgical resection or biopsy is risky when the tumor is located deep in the brain or brainstem. In such cases, a minimally invasive approach to liquid biopsy is beneficial. Cell-free DNA (cfDNA), which directly reflects tumor-specific genetic changes, has attracted attention as a target for liquid biopsy, and blood-based cfDNA monitoring has been demonstrated for other extra-cranial cancers. However, it is still challenging to fully detect CNS tumors derived from cfDNA in the blood, including gliomas, because of the unique structure of the blood–brain barrier. Alternatively, cerebrospinal fluid (CSF) is an ideal source of cfDNA and is expected to contribute significantly to the liquid biopsy of gliomas. Several successful studies have been conducted to detect tumor-specific genetic alterations in cfDNA from CSF using digital PCR and/or next-generation sequencing. This review summarizes the current status of CSF-based cfDNA-targeted liquid biopsy for gliomas. It highlights how the approaches differ from liquid biopsies of other extra-cranial cancers and discusses the current issues and prospects. [ABSTRACT FROM AUTHOR]

Objective To evaluate the value of high-throughput sequencing (HTS) data reanalysis that does not include ERBB2 copy number variation (CNV) analysis, in identifying ERBB2 amplification in patients with colorectal cancer. Methods The HTS data of 252 cases of colorectal cancer diagnosed by pathological biopsy who received peripheral blood cfDNA HTS detection samples were retrospectively analyzed. According to the HTS data of ERBB2 non-amplified samples judged by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), the number of chromosome 17 (Chr17) reads in the total number of reads was calculated the range of the ratio was initially determined as the threshold for prompting ERBB2 amplification. Suspected positive samples were screened according to thresholds and verified by digital PCR, IHC and FISH. Results The proportion of the number of Chr17 reads accounts for the number of total reads in the 89 cases of ERBB2 non-amplified samples determined by IHC and/or FISH ranged from 0.188 to 0.299 (0.239±0.192). Using 0.298(1.25 times the mean) as the threshold indicating ERBB2 amplification, the data of 163 samples were analyzed, of which 7 cases were suspected to be positive, and the ratio ranged from 0.302 to 0.853. Among them, 5 cases were determined to be positive by IHC and/or FISH, and 6 cases were confirmed to be positive by digital PCR. The ratio of the number of Chr17 reads to the number of total reads was positively correlated with the ratio of ERBB2/EIF2C1, and the correlation was good (r²=0.909). Conclusion The high-throughput sequencing data that does not cover the ERBB2 CNV analysis has a certain hint value for ERBB2 amplification in patients with colorectal cancer. [ABSTRACT FROM AUTHOR]

In addition to RUNX1::RUNX1T1 transcript levels, measurable residual disease monitoring using KIT mutant (KITmut) DNA level is reportedly predictive of relapse in t (8; 21) acute myeloid leukemia (AML). However, the usefulness of KITmut transcript levels remains unknown. A total of 202 bone marrow samples collected at diagnosis and during treatment from 52 t (8; 21) AML patients with KITmut (D816V/H/Y or N822K) were tested for KITmut transcript levels using digital polymerase chain reaction. The individual optimal cutoff values of KITmut were identified by performing receiver operating characteristics curve analysis for relapse at each of the following time points: at diagnosis, after achieving complete remission (CR), and after Course 1 and 2 consolidations. The cutoff values were used to divide the patients into the KITmut‐high (KIT_H) group and the KITmut‐low (KIT_L) group. The KIT_H patients showed significantly lower relapse‐free survival (RFS) and overall survival (OS) rates than the KIT_L patients after Course 1 consolidation (p = 0.0040 and 0.021, respectively) and Course 2 consolidation (p = 0.018 and 0.011, respectively) but not at diagnosis and CR. The <3‐log reduction in the RUNX1::RUNX1T1 transcript levels after Course 2 consolidation was an independent adverse prognostic factor for RFS and OS. After Course 2 consolidation, the KIT_H patients with >3‐log reduction in the RUNX1::RUNX1T1 transcript levels (11/45; 24.4%) had similar RFS as that of patients with <3‐log reduction in the RUNX1::RUNX1T1 transcript levels. The combination of KITmut and RUNX1::RUNX1T1 transcript levels after Course 2 consolidation may improve risk stratification in t (8; 21) AML patient with KIT mutation. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) diagnosis still faces challenges with high proportion of bacteriologic test negative incidences worldwide. We assessed the diagnostic value of digital PCR (dPCR) analysis of ultramicro Mycobacterium tuberculosis (M.tb) nucleic acid in CT-guided percutaneous biopsy needle rinse solution (BNRS) for TB. Methods: BNRS specimens were consecutively collected and total DNA was purified. The concentrations of M.tb-specific IS6110 and IS1081 were quantified using droplet dPCR. The diagnostic performances of BNRS-dPCR and its sensitivity in comparison with conventional tests were analyzed. Results: A total of 106 patients were enrolled, 63 of whom were TB (48 definite and 15 clinically suspected TB) and 43 were non-TB. The sensitivity of BNRS IS6110 OR IS1081-dPCR for total, confirmed and clinically suspected TB was 66.7%, 68.8% and 60.0%, respectively, with a specificity of 97.7%. Its sensitivity was higher than that of conventional etiological tests, including smear microscopy, mycobacterial culture and Xpert using sputum and BALF samples. The positive detection rate in TB patients increased from 39.3% for biopsy AFB test alone to 73.2% when combined with BNRS-dPCR, and from 71.4% for biopsy M.tb molecular detection alone to 85.7% when combined with BNRS-dPCR. Conclusion: Our results preliminarily indicated that BNRS IS6110 OR IS1081-dPCR is a feasible etiological test, which has the potential to be used as a supplementary method to augment the diagnostic yield of biopsy and improve TB diagnosis. [ABSTRACT FROM AUTHOR]

Outbreaks of environmentally transmitted parasites require that susceptible hosts encounter transmission stages in the environment and become infected, but we also know that transmission stages can be in the environment without triggering disease outbreaks. One challenge in understanding the relationship between environmental transmission stages and disease outbreaks is that the distribution and abundance of transmission stages outside of their hosts have been difficult to quantify. Thus, we have limited data about how changes in transmission stage abundance influence disease dynamics; moreover, we do not know whether the relationship between transmission stages and outbreaks differs among parasite species. We used digital PCR to quantify the environmental transmission stages of five parasites in six lakes in southeastern Michigan every 2 weeks from June to November 2021. At the same time, we quantified infection prevalence in hosts and host density. Our study focused on eight zooplankton host species (Daphnia spp. and Ceriodaphnia dubia) and five of their parasites from diverse taxonomic groups (bacteria, yeast, microsporidia, and oomycete) with different infection mechanisms. We found that parasite transmission stage concentration increased prior to disease outbreaks for all parasites. However, parasites differed significantly in the relative timing of peaks in transmission stage concentration and infection outbreaks. The "continuous shedder" parasites had transmission stage peaks at the same time as or slightly after the outbreak peaks. In contrast, parasites relying on host death for transmission ("obligate killers") had transmission stage peaks before outbreak peaks. For most parasites, lakes with outbreaks had higher spore concentrations than those without outbreaks, especially once an outbreak began; the exception was for a parasite, Pasteuria ramosa, with very strong genotypic specificity of infection. Overall, our results show that disease outbreaks are tightly linked to transmission stage concentration; outbreaks were preceded by increases in transmission stage concentration in the environment and then were fueled by the production of more transmission stages during the outbreak itself, with concentrations decreasing to pre‐outbreak levels as outbreaks waned. Thus, tracking transmission stages in the environment improves our understanding of the drivers of disease outbreaks and reveals how parasite traits may affect these dynamics. [ABSTRACT FROM AUTHOR]

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders. [ABSTRACT FROM AUTHOR]

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty‐three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next‐generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case‐specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case‐specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case‐specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0–202 days). Two patients with distal metastasis had case‐specific ctDNA in plasma at the time of metastasis. In UTUC, tumor‐specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence. [ABSTRACT FROM AUTHOR]

Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: Multiple myeloma is the second most common malignant hematologic cancer. In approximately 15% of cases, a chromosomal abnormality, t(4;14), leads to uncontrolled production of a protein called NSD2. Different-sized fragments may be missing from the initial segment of this protein, resulting in the creation of three types of breakpoints in the gene encoding the protein. Examining the level of complex (fusion) mRNAs responsible for protein formation might be crucial in the treatment of patients, as lower levels could be associated with a more favorable survival outcome. We have developed a quantitative polymerase chain reaction system capable of detecting measurable residual disease with a sensitivity of up to 1:100,000. Our study also demonstrated that the survival of patients with different breakpoint mRNAs may vary. Among them, those with intermediate-length mRNAs have the worst outcomes. Multiple myeloma (MM) is the second most common hematological malignancy. Approximately 15% of MM patients are affected by the t(4;14) translocation resulting in the IGH::NSD2 fusion transcript. Breakage occurs in three major breakpoint regions within the NSD2 gene (MB4-1, MB4-2, and MB4-3), where MB4-1 leads to the production of full-length protein, while truncated proteins are expressed in the other two cases. Measurable residual disease (MRD) has been conclusively established as a crucial prognostic factor in MM. The IGH::NSD2 fusion transcript can serve as a sensitive MRD marker. Using bone marrow (BM) and peripheral blood (PB) samples from 111 patients, we developed a highly sensitive quantitative real-time PCR (qPCR) and digital PCR (dPCR) system capable of detecting fusion mRNAs with a sensitivity of up to 1:100,000. PB samples exhibited sensitivity three orders of magnitude lower compared to BM samples. Patients with an MB4-2 breakpoint demonstrated significantly reduced overall survival (p = 0.003). Our novel method offers a simple and sensitive means for detecting MRD in a substantial proportion of MM patients. Monitoring may be carried out even from PB samples. The literature lacks consensus regarding survival outcomes among patients with different NSD2 breakpoints. Our data align with previous findings indicating that patients with the MB4-2 breakpoint type tend to exhibit unfavorable overall survival. [ABSTRACT FROM AUTHOR]

Multiplex, digital nucleic acid detections have important biomedical applications, but the multiplexity of existing methods is predominantly achieved using fluorescent dyes or probes, making the detection complicated and costly. Here, we present the StratoLAMP for label-free, multiplex digital loop-mediated isothermal amplification based on visual stratification of the precipitate byproduct. The StratoLAMP designates two sets of primers with different concentrations to achieve different precipitate yields when amplifying different nucleic acid targets. In the detection, deep learning image analysis is used to stratify the precipitate within each droplet and determine the encapsulated targets for nucleic acid quantification. We investigated the effect of the amplification reagents and process on the precipitate generation and optimized the assay conditions. We then implemented a deep-learning image analysis pipeline for droplet detection, achieving an overall accuracy of 94.3%. In the application, the StratoLAMP successfully achieved the simultaneous quantification of two nucleic acid targets with high accuracy. By eliminating the need for fluorescence, StratoLAMP represents a unique concept toward label-free, multiplex nucleic acid assays and an analytical tool with great cost-effectiveness. [ABSTRACT FROM AUTHOR]

This was a pilot study to evaluate the efficacy of digital polymerase chain reaction detection of Demodex in eyelid margin swabs for the diagnosis of Demodex blepharitis. This study aims to explore the possibility of digital polymerase chain reaction detection to improve the diagnostic accuracy of Demodex blepharitis detection. Volunteers were prospectively recruited and classified by experienced doctors into suspected Demodex blepharitis or healthy controls using slit-lamp evaluation of the eyelid margin and an inquiry about symptoms. Three eyelashes from each eyelid were epilated from participants in each group for microscopic observation and mite counting. Then, swabs from the eyelid margins of each eye were collected after the eyelashes were epilated and stored at –80 °C for future DNA extraction and digital polymerase chain reaction detection. The positive or negative results of both methods were compared for diagnostic accuracy, and the Kappa value was also calculated to evaluate their consistency. The accuracy of the digital polymerase chain reaction detection was 71.6% and that of the mite counting method was 75%. Their combined accuracy was improved to 77.3%. The Kappa value of the two methods was 0.505, indicating moderate consistency. Digital polymerase chain reaction detection of Demodex from ocular surface swabs was painless and noninvasive and is a potentially accurate quantitative method available for diagnosing Demodex blepharitis. This method is also complementary to the conventional mite counting method, particularly when a sufficient number of eyelashes cannot be effectively epilated. [ABSTRACT FROM AUTHOR]

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