Detecting Intact Virus Using Exogenous Oligonucleotide Labels.
Detecting Intact Virus Using Exogenous Oligonucleotide Labels.
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DOI：
10.1021/acs.analchem.2c00835
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发表时间：
2022-05
影响因子：
7.4
通讯作者：
T. R. Carey;M. Kozminsky;Jennifer Hall;V. Vargas-Zapata;Kristina M. Geiger;L. Coscoy;Lydia L Sohn
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作者：
T. R. Carey;M. Kozminsky;Jennifer Hall;V. Vargas-Zapata;Kristina M. Geiger;L. Coscoy;Lydia L Sohn
COVID-19大流行揭示了一种新兴病原体如何导致病毒检测需求的突然和急剧增加。检测合并样本可以满足这一需求;然而，逆转录定量聚合酶链反应（RT-qPCR）（金标准）的灵敏度随着合并样本数量的增加而显著降低。在这里，我们介绍了检测完整的病毒的外源核苷酸反应（DIVER），一种方法，定量完整的病毒，是强大的样品稀释。正如使用严重急性呼吸综合征冠状病毒2的两个模型所证明的那样，DIVER首先用外源性寡核苷酸标记膜颗粒，然后在用病毒特异性捕获剂（在这种情况下，血管紧张素转换酶2）功能化的珠子上捕获标记的颗粒，最后使用qPCR定量寡核苷酸标签。使用刺突呈递脂质体和刺突假型慢病毒，我们表明DIVER可以检测1 × 105个脂质体和100个噬斑形成单位的慢病毒，并且可以在合并实验中成功识别阳性样品。总的来说，DIVER非常适合高效的样本汇集和临床验证。
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 × 105 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.