Kline Enos C, Panpradist Nuttada, Hull Ian T, Wang Qin, Oreskovic Amy K, Han Peter D, Starita Lea M, Lutz Barry R
Department of Bioengineering, University of Washingtongrid.34477.33, Seattle, Washington, USA.
Global Health for Women Adolescents and Children, School of Public Health, University of Washingtongrid.34477.33, Seattle, Washington, USA.
Microbiol Spectr. 2022 Aug 31;10(4):e0158321. doi: 10.1128/spectrum.01583-21. Epub 2022 Jun 16.
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 multitarget 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 multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). 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. The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.
在新冠疫情期间，变异谱系的流行率不断上升，由于引物与变异模板之间的错配，有可能干扰分子诊断。即时分子诊断通常缺乏高通量实验室同类产品的完整功能，特别容易受到这类干扰，这可能导致假阴性结果。为应对这一挑战，我们开发了一种强大的环介导等温扩增检测方法，具有单管多重多靶点冗余和内部扩增对照。一种方便且经济高效的靶点特异性荧光检测系统，允许使用适应性探针按信号对扩增进行分组，以便汇总报告新冠病毒靶点扩增情况或区分内部扩增对照。在疫情期间，三种冗余子检测对病毒谱系的引物覆盖范围因检测方法而异，因为它们与武汉 - 1 毒株序列存在差异，但对于所有分析的变异序列，总体覆盖范围仍然很高，最低为 97.4%（关注变异株：伊塔）。在三个实例（德尔塔、伽马、伊塔）中，观察到与三种子检测之一存在高频错配，但由于多靶点冗余，总体覆盖范围仍然很高。当用提取的人类样本进行检测时，多重检测显示出 87%或更高的灵敏度（30 个阳性样本中），对于每个反应含有超过 30 个病毒 RNA 拷贝的样本（21 个阳性样本中）灵敏度为 100%，特异性为 100%（60 个阴性样本中）。这些结果进一步证明，传统实验室方法可在即时检测中加以利用，以实现长期稳健的性能和诊断稳定性。新冠疫情产生了巨大影响，在资源有限的环境中进行分子诊断的能力已成为减轻疾病传播的关键资源。新冠病毒诊断以及其他病毒诊断中的一个挑战是持续的突变，这可能使病毒逃避诊断测试的检测。我们开发了一种检测方法，可在一次检测中检测病毒基因组的多个部分，以减少因突变而漏检病毒的可能性，并且该方法设计得比典型实验室检测更简单、更快，同时保持高灵敏度。这种能力由一种新型荧光探针技术实现，该技术在简单的恒温反应条件下工作。
