Department of Stomatology, the Second People's Hospital of Lianyungang City (Cancer Hospital of Lianyungang), affiliated to Bengbu Medical College, Lianyungang, China.
Front Cell Infect Microbiol. 2022 May 25;12:895261. doi: 10.3389/fcimb.2022.895261. eCollection 2022.
is an important oral pathogen that causes periodontal disease and is difficult to culture under conventional conditions. Therefore, a reliable technique for detecting this pathogenic bacterium is required. Here, isothermal recombinase polymerase amplification (RPA), a new nucleic acid amplification method, was combined with a visualization method based on nanoparticle-based lateral flow strips (LFS) for the rapid detection of . The species-specific 16S rRNA sequence of was used as the target for RPA, and a set of specific primer-probe combinations were designed and screened to amplify the target sequences. As a thermostatic amplification method, the RPA reaction, under optimized conditions, takes only 30 min to complete at a constant temperature (37°C). The amplification reaction products can be detected visually by LFS without any need for special equipment. The RPA-LFS method established for the detection of was shown to be highly specific in distinguishing from other pathogenic organisms by using 20 clinical isolates of and 23 common pathogenic microorganisms. Susceptibility measurements and probit regression analysis were performed with gradient dilutions of genomic DNA. The method was obtained to be highly sensitive, with a detection limit of 9.27 CFU per reaction at 95% probability. By analyzing the gingival sulcus fluid specimens from 130 patients with chronic periodontitis, the results showed that the RPA-LFS method detected 118 positive cases and 12 negative cases of , and the results obtained were consistent with those of a conventional PCR assay. The RPA-LFS method is an efficient, rapid, and convenient diagnostic method that simplifies the tedious process of detecting .
是一种重要的口腔病原体，可引起牙周病，且在常规条件下难以培养。因此，需要一种可靠的技术来检测这种致病细菌。在这里，等温重组酶聚合酶扩增（RPA），一种新的核酸扩增方法，与基于纳米颗粒的侧流条（LFS）的可视化方法相结合，用于快速检测。使用 16S rRNA 序列作为 RPA 的目标，设计并筛选了一组特定的引物-探针组合来扩增目标序列。作为一种恒温扩增方法，在优化条件下，RPA 反应仅需 30 分钟即可在恒定温度（37°C）下完成。扩增反应产物可以通过 LFS 进行目视检测，无需任何特殊设备。通过使用 20 株临床分离株和 23 种常见的致病微生物来区分 ，建立的用于检测 的 RPA-LFS 方法显示出高度的特异性。使用梯度稀释的 基因组 DNA 进行了敏感性测量和概率单位回归分析。该方法具有高度的敏感性，在 95%的概率下检测限为每个反应 9.27 CFU。通过分析 130 名慢性牙周炎患者的龈沟液标本，结果表明 RPA-LFS 方法检测到 118 例阳性和 12 例阴性的 ，与常规 PCR 检测结果一致。RPA-LFS 方法是一种高效、快速、简便的诊断方法，简化了检测 的繁琐过程。
