Effective diagnosis of comorbidities and infectious diseases that present similar symptoms requires point-of-need assays capable of co-detecting and differentiating among multiple co-endemic pathogens to enable timely, precision medicine and effective control measures. We previously developed a two-stage isothermal amplification assay dubbed Penn-RAMP to address this need. Penn-RAMP's first stage comprises a recombinase polymerase amplification (RPA), which amplifies all targets of interest in a single reaction chamber for a short duration. The RPA amplicons are then aliquoted into multiple loop-mediated isothermal amplification (LAMP) reaction chambers, each customized with pre-dried primers to amplify a single target or a group of targets. To enable Penn-RAMP at the point of need, we describe here a self-actuated Penn-RAMP chiplet that accommodates the Penn-RAMP assay. Our chiplet employs temperature-controlled phase change valves and capillary valves to self-aliquot first-stage amplicons into multiple (five) second-stage reaction chambers and to seal these chambers. The functionality of our device is demonstrated by co-detecting plant pathogens. The analytical performance of our chiplet is comparable to that of the benchtop Penn-RAMP assay and surpasses that of standalone LAMP assays. Our self-actuated chiplet can be operated standalone with purified nucleic acids or as the downstream amplification module of a sample preparation cassette.

中文翻译：

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用于两阶段多重核酸扩增测定的自驱动微流控小芯片


有效诊断出现相似症状的合并症和传染病需要能够共同检测和区分多种共同流行病原体的需求点检测，以实现及时、精准的医疗和有效的控制措施。我们之前开发了一种称为 Penn-RAMP 的两阶段等温扩增测定来满足这一需求。Penn-RAMP 的第一阶段包括重组酶聚合酶扩增 （RPA），可在单个反应室中短时间内扩增所有感兴趣的靶标。然后将 RPA 扩增子分装到多个环介导的等温扩增 （LAMP） 反应室中，每个反应室都使用预干燥的引物进行定制，以扩增单个靶标或一组靶标。为了在需要时启用 Penn-RAMP，我们在此处描述了一种适应 Penn-RAMP 测定的自驱动 Penn-RAMP 小芯片。我们的小芯片采用温控相变阀和毛细管阀，将第一阶段扩增子自分装到多个 （5） 个第二阶段反应室中，并密封这些反应室。我们设备的功能通过共同检测植物病原体来证明。我们的小芯片的分析性能与台式 Penn-RAMP 检测相当，并超过了独立的 LAMP 检测。我们的自驱动小芯片可以与纯化的核酸一起独立运行，也可以作为样品制备盒的下游扩增模块。