Physical Chemistry and Applied Spectroscopy, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Biosecurity and Public Health, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Biosensors (Basel). 2021 Sep 30;11(10):367. doi: 10.3390/bios11100367.
Detection methods that do not require nucleic acid amplification are advantageous for viral diagnostics due to their rapid results. These platforms could provide information for both accurate diagnoses and pandemic surveillance. Influenza virus is prone to pandemic-inducing genetic mutations, so there is a need to apply these detection platforms to influenza diagnostics. Here, we analyzed the Fast Evaluation of Viral Emerging Risks (FEVER) pipeline on ultrasensitive detection platforms, including a waveguide-based optical biosensor and a flow cytometry bead-based assay. The pipeline was also evaluated in silico for sequence coverage in comparison to the U.S. Centers for Disease Control and Prevention's (CDC) influenza A and B diagnostic assays. The influenza FEVER probe design had a higher tolerance for mismatched bases than the CDC's probes, and the FEVER probes altogether had a higher detection rate for influenza isolate sequences from GenBank. When formatted for use as molecular beacons, the FEVER probes detected influenza RNA as low as 50 nM on the waveguide-based optical biosensor and 1 nM on the flow cytometer. In addition to molecular beacons, which have an inherently high background signal we also developed an exonuclease selection method that could detect 500 pM of RNA. The combination of high-coverage probes developed using the FEVER pipeline coupled with ultrasensitive optical biosensors is a promising approach for future influenza diagnostic and biosurveillance applications.
由于检测结果快速，因此不需要核酸扩增的检测方法在病毒诊断方面具有优势。这些平台可以为准确诊断和大流行监测提供信息。流感病毒容易发生引起大流行的遗传突变，因此有必要将这些检测平台应用于流感诊断。在这里，我们分析了基于超灵敏检测平台的快速评估病毒新兴风险 (FEVER) 管道，包括基于波导的光学生物传感器和基于流式细胞术珠子的检测。还通过计算机模拟对该管道与美国疾病控制与预防中心 (CDC) 的流感 A 和 B 诊断检测的序列覆盖范围进行了评估。与 CDC 的探针相比，流感 FEVER 探针设计对不匹配碱基具有更高的容忍度，并且 FEVER 探针总体上对 GenBank 中流感分离株序列的检测率更高。当格式化为用作分子信标时，FEVER 探针在基于波导的光学生物传感器上可低至 50 nM 检测到流感 RNA，在流式细胞仪上可低至 1 nM 检测到流感 RNA。除了具有固有高背景信号的分子信标之外，我们还开发了一种外切酶选择方法，该方法可检测 500 pM 的 RNA。使用 FEVER 管道开发的高覆盖率探针与超灵敏光学生物传感器的结合是未来流感诊断和生物监测应用的有前途的方法。
