Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.

Manifold reaching paradigm: how do we handle target redundancy?

How the central nervous system coordinates the many intrinsic degrees of freedom of the musculoskeletal system is a recurrent question in motor control. Numerous studies addressed it by considering redundant reaching tasks such as point-to-point arm movements, for which many joint trajectories and muscle activations are usually compatible with a single goal. There exists, however, a different, extrinsic kind of redundancy that is target redundancy. Many times, indeed, the final point to reach is neither specified nor unique. In this study, we aim to understand how the central nervous system tackles such an extrinsic redundancy by considering a reaching-to-a-manifold paradigm, more specifically an arm pointing to a long vertical bar. In this case, the endpoint is not defined a priori and, therefore, subjects are free to choose any point on the bar to successfully achieve the task. We investigated the strategies used by subjects to handle this presented choice. Our results indicate both intersubject and intertrial consistency with respect to the freedom provided by the task. However, the subjects' behavior is found to be more variable than during classical point-to-point reaches. Interestingly, the average arm trajectories to the bar and the structure of intertrial endpoint variations could be explained via stochastic optimal control with an energy/smoothness expected cost and signal-dependent motor noise. We conclude that target redundancy is first overcome during movement planning and then exploited during movement execution, in agreement with stochastic optimal feedback control principles, which illustrates how the complementary problems of goal and movement selection may be resolved at once.