1. Digital PCR outperforms quantitative real-time PCR for the detection and quantification of major periodontal pathobiontsHaris Munjaković, Katja Povšič, Mario Poljak, Katja Seme, Rok Gašperšič, Lucijan Skubic, 2025, izvirni znanstveni članek Povzetek: Background: This study comparatively evaluated the analytical and diagnostic performance of a multiplex digital polymerase-chain reaction (dPCR) assay and a quantitative real-time PCR (qPCR) for the simultaneous detection and quantification of periodontal pathobionts: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. Materials and methods: Subgingival plaque samples from 20 periodontitis patients and 20 periodontally healthy controls were analyzed. Several analytical parameters of the dPCR assay, optimized using DNA standards, were assessed versus qPCR: dynamic range linearity, precision, accuracy, prevalence, sensitivity, specificity, and concordance. The statistical analyses included Mann-Whitney U test, Wilcoxon test, McNemar's test, and Bland-Altman plots. Results: dPCR showed high linearity (R2 > 0.99) and lower intra-assay variability (median CV%: 4.5%) than qPCR (p = 0.020), with comparable accuracy and agreement. dPCR demonstrated superior sensitivity, detecting lower bacterial loads, particularly for P. gingivalis and A. actinomycetemcomitans. The Bland-Altman plots highlighted good agreement at medium/high loads but discrepancies at low concentrations (< 3 log10Geq/mL), resulting in qPCR false negatives and a 5-fold underestimation of the prevalence of A. actinomycetemcomitans in periodontitis patients. High concordance between the assays was observed for F. nucleatum across both study groups. Conclusions: dPCR outperformed qPCR for quantifying periodontal pathobionts and had superior sensitivity and precision, making it particularly effective in detecting low-level bacterial loads.
Ključne besede: digital PCR, oral microbiology, periodontal disease, quantitative real-time PCR, subgingival plaque
Objavljeno v DiRROS: 15.04.2026; Ogledov: 165; Prenosov: 106
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2. Absolute quantification of microRNA miR-875-5p in temporal artery biopsies and its biomarker potential for giant cell arteritisLuka Bolha, Elvisa Smajlović, Alojzija Hočevar, 2026, drugi znanstveni članki Povzetek: Objectives: To perform absolute quantification of miR-875-5p expression in temporal artery biopsies (TABs) of patients with giant cell arteritis (GCA), associate miR-875-5p copy number with histological and clinical characteristics of patients with GCA, and evaluate the diagnostic value of absolute copy number of miR-875-5p in GCA. Methods: The study included formalin-fixed, paraffin-embedded TABs of 45 treatment-naïve clinically proven patients with GCA, and 19 non-GCA controls. Of the included patients with GCA, 29 had histologically positive and 16 histologically negative TABs. Expression of miR-875-5p was assessed through utilization of quantitative real-time PCR (qPCR) and absolute quantification by digital PCR (dPCR). Results: We determined significantly higher absolute copy number of miR-875-5p in histologically positive TABs of patients with GCA, compared to histologically negative TABs of GCA and non-GCA patients, which significantly correlated with the majority of TAB histopathological parameters and several clinical characteristics of patients with GCA. Notably, we showed a good diagnostic performance of absolute copy number of miR-875-5p in discriminating patients and controls, depending on the extent of vessel wall inflammation and remodeling in affected temporal arteries. Conclusion: Our study revealed that absolute quantification of miR-875-5p expression holds the potential to serve as a supporting biomarker in assessing vessel wall inflammation and remodeling in patients with GCA. Moreover, our results indicate the applicability of absolute quantification by dPCR in detecting low-abundance miRNAs in GCA-affected arterial tissue, whose limiting amounts hinder the utilization of classical qPCR.
Ključne besede: biomarker, digital PCR, giant cell arteritis, microRNA, temporal artery biopsy
Objavljeno v DiRROS: 25.03.2026; Ogledov: 221; Prenosov: 127
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3. Simple, fast, reliable: multiplex digital PCR quantification of 19 genetically modified soybean eventsAmadej Jelenčič, Dejan Štebih, Tina Demšar, David Dobnik, 2026, izvirni znanstveni članek Povzetek: Plant genetic engineering represents an important aspect of modern agriculture, and new geneticallymodified (GM) crop varieties are entering the market on a regular basis. This necessitates thedevelopment of high throughput multi-target analytical methods to detect and quantify theirpresence for regulatory compliance. In this study, we present a multiplex dPCR method for discrimi-native quantification of 19 GM soybean events and the lectin (Le1) endogene on a nanowell plate-based all-in-one dPCR system. The method consists of four 5-plex assays, taking advantage of theplatform’s multiple fluorescence detection channels. The assays complied with the minimum perfor-mance requirements in terms of specificity, trueness, precision, sensitivity and dynamic range,making them suitable for use in routine detection and quantification of GM crops. This methodrepresents the most comprehensive multi-target GM soybean quantification approach to date with-out the need for prior screening and features a simplified workflow, making it suitable for widespreadadoption. Our study sets a precedent for rapid and straightforward development of multiplex dPCRGM crop quantification assays to address the evolving demands of regulatory monitoring.
Ključne besede: genetically modified (GM) crops, transgenic, soybean, Glycine max (L.) merr, quantification, digital PCR (dPCR), multiplex, multi-target
Objavljeno v DiRROS: 19.03.2026; Ogledov: 245; Prenosov: 183
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4. Assessing nuclear versus mitochondrial cell-free DNA (cfDNA) by qRT-PCR and droplet digital PCR using a piglet model of perinatal asphyxiaMarie Bitenc, Benedicte Grebstad Tune, Maria Melheim, Monica Atneosen‑Åsegg, Polona Rajar, 2023, izvirni znanstveni članek Povzetek: Background: Since the discovery more than half a century ago, cell-free DNA (cfDNA) has become an attractive objective in multiple diagnostic, prognostic, and monitoring settings. However, despite the increasing number of cfDNA applications in liquid biopsies, we still lack a comprehensive understanding of the nature of cfDNA including optimal assessment. In the presented study, we continued testing and validation of common techniques for cfDNA extraction and quantification (qRTPCR or droplet digital PCR) of nuclear- and mitochondrial cfDNA (ncfDNA and mtcfDNA) in blood, using a piglet model of perinatal asphyxia to determine potential temporal and quantitative changes at the levels of cfDNA. Methods and Results: Newborn piglets (n=19) were either exposed to hypoxia (n=11) or were part of the sham-operated control group (n=8). Blood samples were collected at baseline (=start) and at the end of hypoxia or at 40–45 min for the sham-operated control group. Applying the qRT-PCR method, ncfDNA concentrations in piglets exposed to hypoxia revealed an increasing trend from 7.1 ng/ml to 9.5 ng/ml for HK2 (hexokinase 2) and from 4.6 ng/ml to 7.9 ng/ml for β-globulin, respectively, whereas the control animals showed a more balanced profile. Furthermore, median levels of mtcfDNA were much higher in comparison to ncfDNA, but without significant differences between intervention versus the control group. Conclusions: Both, qRT-PCR and the droplet digital PCR technique identified overall similar patterns for the concentration changes of cfDNA; but, the more sensitive digital PCR methodology might be required to identify minimal responses.
Ključne besede: cell-free DNA, digital PCR, mitochondrial cfDNA, nuclear cfDNA, perinatal asphyxia, qRT-PCR
Objavljeno v DiRROS: 25.02.2026; Ogledov: 354; Prenosov: 182
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5. Somatic mutation detection in tumor tissue and matched cell-free DNA using PCR-based methods in pancreatic cancer patients undergoing upfront resectionHana Zavrtanik, David Badovinac, Tanja Blagus, Katja Goričar, Branislava Ranković, Alenka Matjašič, Andrej Zupan, Aleš Tomažič, Vita Dolžan, 2025, izvirni znanstveni članek Povzetek: Somatic mutations in KRAS and TP53 are among the most common genetic alterations in pancreatic ductal adenocarcinoma (PDAC). Advances in PCR-based technologies now enable the detection of these mutations in tumor tissue and cell-free DNA (cfDNA), providing a minimally invasive approach to assess tumor burden. However, in resectable PDAC, circulating tumor DNA (ctDNA) may represent less than 0.1% of total cfDNA, requiring highly sensitive detection methods. The aim of our study was to assess two PCR-based assays—competitive allele-specific PCR (castPCR) and digital PCR (dPCR)—for detecting selected somatic mutations in tumor tissue, cfDNA, and extracellular vesicle-associated DNA (EV-DNA) from plasma. Matched primary tumor and preoperative plasma samples were collected from 50 patients undergoing upfront resection for PDAC. CastPCR was used for detecting selected KRAS, TP53, SMAD4, and CDKN2A mutations in tumor DNA. Additionally, dPCR was used to analyze KRAS and TP53 mutations in tumor DNA as well as cfDNA and EV-DNA. The concordance between both platforms was 71.4% for KRAS p.G12D and 58.3% for the analysis of TP53 p.R273H mutations in tumor tissue. However, dPCR detected these mutations in an additional 28.6% and 39.6% of samples, respectively. In cfDNA, dPCR identified KRAS p.G12D in 10.2% and TP53 p.R273H in 2.0% of samples. Mutation detection in EV-DNA was limited by low DNA yield. Both platforms proved effective for tumor DNA analysis, with dPCR offering greater sensitivity. Somatic mutation detection from liquid biopsy using dPCR further supports its potential utility in the preoperative setting.
Ključne besede: somatic mutation detection, cell-free DNA, liquid biopsy, digital PCR, pancreatic cancer
Objavljeno v DiRROS: 08.01.2026; Ogledov: 530; Prenosov: 230
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6. Multiplex quantification of 19 GM soybean lines using digital PCRAmadej Jelenčič, Dejan Štebih, Tina Demšar, David Dobnik, 2026, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Povzetek: The European Union (EU) imposes strict regulations on the presence of genetically modified (GM) material in food and feed, requiring thorough testing of samples for various GM lines. Although traditional quantitative real-time PCR (qPCR) methods are sensitive and robust, they are not cost-effective for managing large numbers of GM events due to their limited multiplexing capabilities. Conversely, digital PCR (dPCR) is capable of robust quantitative multiplexing in addition to other benefits such as absolute quantification and better tolerance of PCR inhibitors. In this context, we present a protocol for multiplex quantification of 19 GM soybean lines using dPCR as an improvement over the currently used simplex qPCR approach. This method enables simple and robust quantification of common GM soybean lines with a relatively low number of reactions.
Ključne besede: genetically modified soybean, GMO, multiplexing, food and feed, food regulation, digital PCR (dPCR)
Objavljeno v DiRROS: 26.11.2025; Ogledov: 1026; Prenosov: 172
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7. Digital PCR-based genotyping: a precision approach to HCMV drug resistanceMojca Milavec, Tašja Cvelbar, Alexandra Bogožalec Košir, 2025, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Povzetek: The genotyping workflow described uses digital PCR (dPCR) to detect and quantify drug resistance mutations in human cytomegalovirus (HCMV). The method focuses on the detection and quantification of three common mutations in the UL97 gene at codons 460, 594, and 595, which are responsible for the majority of ganciclovir-resistant clinical isolates. The dPCR approach offers high sensitivity and accuracy, making it suitable for routine testing as well as a reference measurement procedure for external quality assessment schemes. The workflow includes several key steps: DNA isolation, preparation of the dPCR reaction mixture, partitioning, thermocycling, and data analysis. This method improves the detection capabilities of HCMV drug resistance and provides a robust and efficient tool for clinical and research applications.
Ključne besede: digital PCR, human cytomegalovirus, antimicrobial drug resistance, mutations
Objavljeno v DiRROS: 25.09.2025; Ogledov: 510; Prenosov: 112
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8. Digital PCR genotyping of pepino mosaic virusAmadej Jelenčič, David Dobnik, Alexandra Bogožalec Košir, Nataša Mehle, 2025, samostojni znanstveni sestavek ali poglavje v monografski publikaciji Povzetek: Pepino mosaic virus (PepMV) is a plant pathogen causing significant economic losses in tomato production. Sensitive, reliable, and robust detection methods are crucial for containing the spread of PepMV and reducing its damaging effects. Digital PCR (dPCR) presents several advantages to conventional real-time quantitative PCR (qPCR), including absolute quantification ability, robust quantitative multiplexing capabilities, and straightforward result analysis. Furthermore, dPCR is especially suitable for analysis of complex samples due to its remarkable tolerance to PCR inhibitors, which makes it a promising method for plant virus genotyping. In this chapter, we present two protocols for PepMV genotyping and quantification using one-step reverse transcription digital PCR (RT-dPCR). The first protocol outlines four simplex assays, while the second describes two duplex assays for precise and comprehensive genotyping of PepMV variants.
Ključne besede: plant viruses, detection, quantification, Pepino mosaic virus, PepMV, digital PCR, dPCR, RT-dPCR, duplex
Objavljeno v DiRROS: 08.09.2025; Ogledov: 750; Prenosov: 106
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9. One-step RT-droplet digital PCR : a breakthrough in the quantification of waterborne RNA virusesNejc Rački, Dany Morisset, Ion Gutiérrez-Aguirre, Maja Ravnikar, 2014, izvirni znanstveni članek Povzetek: Water contamination by viruses has an increasing worldwide impact on human health, and has led to requirements for accurate and quantitative molecular tools. Here, we report the first one-step reverse-transcription droplet digital PCR-based absolute quantification of a RNA virus (rotavirus) in different types of surface water samples. This quantification method proved to be more precise and more tolerant to inhibitory substances than the benchmarking reverse-transcription real-time PCR (RT-qPCR), and needs no standard curve. This new tool is fully amenable for the quantification of viruses in the particularly low concentrations usually found in water samples.
Ključne besede: waterborne virus, quantification, droplet digital PCR, real-time PCR, digital PCR
Objavljeno v DiRROS: 04.03.2025; Ogledov: 1097; Prenosov: 716
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10. Quantitative analysis of food and feed samples with droplet digital PCRDany Morisset, Dejan Štebih, Mojca Milavec, Kristina Gruden, Jana Žel, 2013, izvirni znanstveni članek Povzetek: In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.
Ključne besede: droplet digital PCR, ddPCR, genetically modified organisms
Objavljeno v DiRROS: 04.03.2025; Ogledov: 873; Prenosov: 790
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