UC Berkeley-UC San Francisco Graduate Program in Bioengineering, University of California, Berkeley, 306 Stanley Hall, Berkeley, California 94720, United States.
California Institute for Quantitative Biosciences, University of California, Berkeley, 174 Stanley Hall, Berkeley, California 94720, United States.
Anal Chem. 2022 May 31;94(21):7619-7627. doi: 10.1021/acs.analchem.2c00835. Epub 2022 May 18.
The COVID-19 pandemic has revealed how an emerging pathogen can cause a sudden and dramatic increase in demand for viral testing. Testing pooled samples could meet this demand; however, the sensitivity of reverse transcription quantitative polymerase chain reaction (RT-qPCR), the gold standard, significantly decreases with an increasing number of samples pooled. Here, we introduce detection of intact virus by exogenous-nucleotide reaction (DIVER), a method that quantifies intact virus and is robust to sample dilution. As demonstrated using two models of severe acute respiratory syndrome coronavirus 2, DIVER first tags membraned particles with exogenous oligonucleotides, then captures the tagged particles on beads functionalized with a virus-specific capture agent (in this instance, angiotensin-converting enzyme 2), and finally quantifies the oligonucleotide tags using qPCR. Using spike-presenting liposomes and spike-pseudotyped lentivirus, we show that DIVER can detect 1 × 10 liposomes and 100 plaque-forming units of lentivirus and can successfully identify positive samples in pooling experiments. Overall, DIVER is well positioned for efficient sample pooling and clinical validation.
新冠疫情揭示了一种新出现的病原体如何导致病毒检测需求的突然和显著增加。聚合样本检测可以满足这一需求；然而，作为金标准的逆转录定量聚合酶链反应 (RT-qPCR) 的灵敏度随着聚合样本数量的增加而显著降低。在这里，我们介绍了通过外源核苷酸反应 (DIVER) 检测完整病毒的方法，该方法可以定量完整病毒，并且对样品稀释具有很强的稳健性。我们使用两种严重急性呼吸综合征冠状病毒 2 模型进行了演示，DIVER 首先用外源寡核苷酸标记膜颗粒，然后用带有病毒特异性捕获剂（在本例中为血管紧张素转换酶 2）的珠子捕获标记的颗粒，最后使用 qPCR 定量寡核苷酸标记。我们使用带有刺突的脂质体和带有刺突的慢病毒假型病毒展示，表明 DIVER 可以检测到 1×10 个脂质体和 100 个慢病毒噬菌斑形成单位，并且可以成功识别聚合实验中的阳性样本。总的来说，DIVER 非常适合高效的样本聚合和临床验证。
