Digital PCR outperforms quantitative real-time PCR for the detection and quantification of major periodontal pathobionts.

IF 5.5 2区 医学 Q2 MICROBIOLOGY
Journal of Oral Microbiology Pub Date : 2025-07-23 eCollection Date: 2025-01-01 DOI:10.1080/20002297.2025.2537439
Haris Munjaković, Katja Povšič, Mario Poljak, Katja Seme, Rok Gašperšič, Lucijan Skubic
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引用次数: 0

Abstract

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.

数字PCR优于定量实时PCR检测和定量主要牙周病原体。
背景:本研究比较了多重数字聚合酶链反应(dPCR)法和实时荧光定量PCR (qPCR)法同时检测和定量牙周病原菌:牙龈卟啉单胞菌、放线菌聚集杆菌和核梭杆菌的分析和诊断性能。材料与方法:对20例牙周炎患者和20例牙周健康对照者的龈下菌斑进行分析。使用DNA标准优化的dPCR检测的几个分析参数与qPCR进行了比较:动态范围线性、精密度、准确性、患病率、敏感性、特异性和一致性。统计分析包括Mann-Whitney U检验、Wilcoxon检验、McNemar检验和Bland-Altman图。结果:与qPCR相比,dPCR具有较高的线性(R2为0.99)和较低的测定内变异性(中位CV%: 4.5%) (p = 0.020),具有相当的准确性和一致性。dPCR表现出更高的灵敏度,检测到较低的细菌负荷,特别是牙龈假单胞菌和放线菌comitans。Bland-Altman图强调了在中/高负荷下的良好一致性,但在低浓度(10Geq/mL)下存在差异,导致qPCR假阴性,并低估了牙周炎患者放线菌comitans患病率的5倍。在两个研究组中观察到核梭菌检测结果的高度一致性。结论:dPCR在牙周病原菌定量方面优于qPCR,具有更高的灵敏度和精密度,尤其适用于检测低水平细菌负荷。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
8.00
自引率
4.40%
发文量
52
审稿时长
12 weeks
期刊介绍: As the first Open Access journal in its field, the Journal of Oral Microbiology aims to be an influential source of knowledge on the aetiological agents behind oral infectious diseases. The journal is an international forum for original research on all aspects of ''oral health''. Articles which seek to understand ''oral health'' through exploration of the pathogenesis, virulence, host-parasite interactions, and immunology of oral infections are of particular interest. However, the journal also welcomes work that addresses the global agenda of oral infectious diseases and articles that present new strategies for treatment and prevention or improvements to existing strategies. Topics: ''oral health'', microbiome, genomics, host-pathogen interactions, oral infections, aetiologic agents, pathogenesis, molecular microbiology systemic diseases, ecology/environmental microbiology, treatment, diagnostics, epidemiology, basic oral microbiology, and taxonomy/systematics. Article types: original articles, notes, review articles, mini-reviews and commentaries
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