Quantification of the HIV viral reservoir is critical to understanding HIV latency, advancing patient care and ultimately achieving a cure. To quantify the reservoir, a new metric was recently introduced, which quantified cells carrying multiply spliced HIV RNA. The developed assay, Tat/rev Induced Limiting Dilution Assay (TILDA), enables quantification of cells containing multiply-spliced HIV RNA events as an indicator of reservoir size. Due to TILDA's reliance on a limiting dilution format paired with the rarity of target events, numerous individual reactions are required to obtain a single endpoint. The current assay embodiment uses a whole cell input to detect target RNA sequences without the traditional preceding nucleic acid purification steps. Thus, while the direct measurement of target events from whole cells significantly streamlines the workflow, there is a cost in sensitivity and assay throughput. Here, we apply a new technique for rapid RNA isolation, Exclusion-Based Sample Preparation, to TILDA, with the goal of alleviating these limitations without significantly adding to the workflow. By combining TILDA with multiplexed RNA extraction enabled by exclusion-based sample preparation, assay sensitivity and capacity are improved while maintaining assay simplicity, advancements that could facilitate eventual clinical implementation in detecting rare events in patients.

中文翻译：

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RNA介导的TILDA可改善细胞容量并增强对多重剪接的HIV RNA的检测

HIV病毒库的量化对于了解HIV潜伏期，推进患者护理并最终实现治愈至关重要。为了量化储库，最近引入了一种新的度量标准，该度量对携带多重剪接的HIV RNA的细胞进行了量化。开发的检测方法，Tat / rev诱导极限稀释检测法（TILDA），能够量化包含多重剪接的HIV RNA事件的细胞，作为储库大小的指标。由于TILDA依赖于有限的稀释形式以及很少的靶标事件，因此需要许多单独的反应才能获得单个终点。当前的测定实施方案使用全细胞输入来检测靶RNA序列，而无需传统的先前核酸纯化步骤。因此，尽管直接测量全细胞中的靶标事件可显着简化工作流程，但灵敏度和检测通量却要付出一定的代价。在这里，我们将一种用于快速RNA分离的新技术，即基于排阻的样品制备，应用于TILDA，目的是减轻这些限制，而又不会显着增加工作流程。通过将TILDA与通过基于排阻的样品制备实现的多重RNA提取相结合，可提高测定的灵敏度和能力，同时保持测定的简便性，这些进展可促进最终临床实施以检测患者的罕见事件。目的是在不显着增加工作流程的情况下减轻这些限制。通过将TILDA与通过基于排阻的样品制备实现的多重RNA提取相结合，可提高测定的灵敏度和能力，同时保持测定的简便性，这些进展可促进最终临床实施以检测患者的罕见事件。目的是在不显着增加工作流程的情况下减轻这些限制。通过将TILDA与通过基于排阻的样品制备实现的多重RNA提取相结合，可提高测定的灵敏度和能力，同时保持测定的简便性，这些进展可促进最终临床实施以检测患者的罕见事件。