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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: 206; Prenosov: 156
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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: 247; Prenosov: 145
Celotno besedilo (1,11 MB)
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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: 292; Prenosov: 235
Celotno besedilo (1,03 MB)
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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: 396; Prenosov: 197
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Ključne besede: varstvo gozdov, žuželke, identifikacija, diagnostični protokol, molekularna analiza, ekstrakcija DNA, PCR v realnem času, verifikacija
Objavljeno v DiRROS: 16.01.2026; Ogledov: 351; Prenosov: 0
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