Department of Laboratory Medicine, Huadong Hospital Affiliated to Fudan University, Shanghai, China.
Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital Affiliated to Fudan University, Shanghai, China.
Front Cell Infect Microbiol. 2023 Sep 29;13:1267288. doi: 10.3389/fcimb.2023.1267288. eCollection 2023.
This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of directly from the clinical non-invasive oral samples.
The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted gene, semi-quantitative gene and 10 virulence genes of . Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the loads in different clinical samples were further compared. The mixed plasmids of virulence genes , and were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of -positive patients was compared.
The non-invasive HMGA was highly specific for detection of all 12 targets of and human internal reference gene , and the sensitivity to all target genes could reach 10 copies/μL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (>0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of in oral samples. Moreover, by detecting peak area levels of , it was confirmed that the loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (<0.05). We also found that 45.0% (91/202) of patients had different virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (<0.05).
The non-invasive HMGA proved to be a reliable method for the rapid identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of .
本研究建立了一种高通量多重基因检测分析方法（HMGA），用于快速鉴定、半定量和毒力分析，直接从临床非侵入性口腔样本中。
本研究从 2021 年至 2022 年在上海采集了 242 名患者的胃黏膜和口腔样本。所有样本均通过常规临床检测进行检测，并对结果不一致的样本进行 Sanger 测序。设计并优化了一种新的多重 PCR 检测方法，该方法涉及针对基因、基因和 10 个毒力基因的荧光染料标记特异性引物，可在 4 小时内提供结果。通过模拟阳性口腔样本的 10 倍系列稀释，进行半定量检测，并进一步比较不同临床样本中的载量。使用混合质粒的毒力基因、和，评估不同基因型的性能。比较了幽门螺杆菌阳性患者胃黏膜、唾液、漱口液和牙菌斑中 10 个毒力基因的一致性。
非侵入性 HMGA 对所有 12 个目标基因和人类内参基因的检测具有高度特异性，对所有目标基因的敏感性均可达到 10 拷贝/μL。与常规临床检测和测序相比，非侵入性 HMGA 对口腔样本中直接检测的敏感性、特异性、准确性、PPV、NPV 和 Kappa 系数均具有较高水平（>0.98）。此外，通过检测基因的峰面积水平，证实胃黏膜中的载量明显高于三种口腔样本（<0.05）。我们还发现，45.0%（91/202）的患者在不同口腔样本中具有不同的毒力基因。唾液和胃黏膜中每个毒力基因阳性检测率的一致性均超过 78%（<0.05）。
非侵入性 HMGA 被证明是一种可靠的方法，可用于快速鉴定、半定量和直接检测口腔样本中的 10 个毒力基因，为非侵入性检测提供了新的思路。
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