Institute of Microbiology and Virology, Brandenburg Medical School Theodor Fontane, Universitätsplatz 1, 01968 Senftenberg, Germany.
Department of Virology, University Medical Center, Kreuzbergring 57, 37075 Göttingen, Germany.
Anal Chem. 2024 Feb 27;96(8):3267-3275. doi: 10.1021/acs.analchem.3c02985. Epub 2024 Feb 15.
Nosocomial-associated diarrhea due to infection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B ( and ). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 strains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 μL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 μL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.
医院相关性腹泻由 感染（CDI）引起，在样本预培养后，通过酶免疫分析检测毒素或通过毒素基因核酸扩增来诊断。快速和直接的诊断对于靶向治疗以预防严重病例和复发非常重要。我们开发了两种针对毒素基因 A 和 B（和）的单重和一种一步式双重荧光 15 分钟等温重组聚合酶扩增（RPA）检测。此外，我们将单重 RPA 适配到 3D 打印微流控反应器设备上。使用 DNA 标准和 20 株具有不同毒素型的 DNA 提取物来确定分析灵敏度。用 19 株梭菌和胃肠道细菌菌株来确定分析特异性。在 50 μL 体积中，将单重分析适配为双重分析需要优化引物和探针浓度。通过将体积减少四分之一（12.4 μL），建立了用于 3D 打印微反应器的体积减少。RPA 的混合被证实是优化分析灵敏度的关键。在双管格式和单重 3D 打印微反应器格式中，检测限（LOD）范围为 119 至 1411 DNA 分子，检测结果相似。双重 RPA 允许同时检测对 CDI 及时和可靠诊断很重要的两种毒素。3D 打印反应室可以开发成一种微流控即时检测系统，用于床边检测。
