() infection correlates closely with gastric diseases such as gastritis, ulcers, and cancer, influencing more than half of the world's population. Establishing a rapid, precise, and automated platform for diagnosis is an urgent clinical need and would significantly benefit therapeutic intervention. Recombinase polymerase amplification (RPA)-CRISPR recently emerged as a promising molecular diagnostic assay due to its rapid detection capability, high specificity, and mild reaction conditions. In this work, we adapted the RPA-CRISPR assay on a digital microfluidics (DMF) system for automated detection and genotyping. The system can achieve multi-target parallel detection of nucleotide conservative genes () and virulence genes ( and ) across different samples within 30 min, exhibiting a detection limit of 10 copies/rxn and no false positives. We further conducted tests on 80 clinical saliva samples and compared the results with those derived from real-time quantitative polymerase chain reaction, demonstrating 100% diagnostic sensitivity and specificity for the RPA-CRISPR/DMF method. By automating the assay process on a single chip, the DMF system can significantly reduce the usage of reagents and samples, minimize the cross-contamination effect, and shorten the reaction time, with the additional benefit of losing the chance of experiment failure/inconsistency due to manual operations. The DMF system together with the RPA-CRISPR assay can be used for early detection and genotyping of with high sensitivity and specificity, and has the potential to become a universal molecular diagnostic platform.

中文翻译：

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利用数字微流控技术对幽门螺杆菌进行快速多靶点基因分型

()感染与胃炎、溃疡和癌症等胃部疾病密切相关，影响着世界一半以上的人口。建立快速、精确、自动化的诊断平台是临床的迫切需求，并将显着有利于治疗干预。重组酶聚合酶扩增（RPA）-CRISPR由于其快速检测能力、高特异性和温和的反应条件而最近成为一种有前途的分子诊断检测方法。在这项工作中，我们在数字微流体 (DMF) 系统上采用了 RPA-CRISPR 检测，以进行自动检测和基因分型。该系统可在30分钟内实现不同样本的核苷酸保守基因()和毒力基因(和)的多靶点并行检测，检测限为10拷贝/rxn，无假阳性。我们进一步对 80 个临床唾液样本进行了测试，并将结果与​​实时定量聚合酶链式反应得出的结果进行了比较，证明 RPA-CRISPR/DMF 方法具有 100% 的诊断灵敏度和特异性。通过在单芯片上实现检测过程的自动化，DMF系统可以显着减少试剂和样品的使用，最大限度地减少交叉污染效应，并缩短反应时间，并具有减少由于实验失败/不一致的机会的额外好处到手动操作。 DMF系统与RPA-CRISPR检测一起可用于具有高灵敏度和特异性的早期检测和基因分型，并有潜力成为通用的分子诊断平台。