Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, TX 78712, USA.
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Viruses. 2018 Dec 14;10(12):714. doi: 10.3390/v10120714.
We have developed a generalizable "smart molecular diagnostic" capable of accurate point-of-care (POC) detection of variable nucleic acid targets. Our isothermal assay relies on multiplex execution of four loop-mediated isothermal amplification reactions, with primers that are degenerate and redundant, thereby increasing the breadth of targets while reducing the probability of amplification failure. An easy-to-read visual answer is computed directly by a multi-input Boolean OR logic gate (gate output is true if either one or more gate inputs is true) signal transducer that uses degenerate strand exchange probes to assess any combination of amplicons. We demonstrate our methodology by using the same assay to detect divergent Asian and African lineages of the evolving Zika virus (ZIKV), while maintaining selectivity against non-target viruses. Direct analysis of biological specimens proved possible, with crudely macerated ZIKV-infected mosquitoes being identified with 100% specificity and sensitivity. The ease-of-use with minimal instrumentation, broad programmability, and built-in fail-safe reliability make our smart molecular diagnostic attractive for POC use.
我们开发了一种可推广的“智能分子诊断”方法，能够在现场（POC）准确检测可变核酸靶标。我们的等温测定法依赖于四个环介导的等温扩增反应的多重执行，使用的引物是简并和冗余的，从而增加了靶标的广度，同时降低了扩增失败的概率。一个易于读取的视觉答案是由一个多输入布尔或逻辑门（如果一个或多个门输入为真，则门输出为真）信号转换器直接计算得出的，该信号转换器使用简并链交换探针来评估任何组合的扩增子。我们通过使用相同的测定法来检测不断进化的寨卡病毒（ZIKV）的亚洲和非洲发散谱系，同时保持对非靶病毒的选择性，来证明我们的方法。直接分析生物样本是可能的，用粗磨碎的感染寨卡病毒的蚊子进行鉴定，具有 100%的特异性和敏感性。这种智能分子诊断具有易于使用、最小化仪器、广泛的可编程性和内置的故障安全可靠性，非常适合现场使用。
