Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
Biomedical Engineering Department, Jordan University of Science and Technology, Irbid, Jordan.
Nat Protoc. 2021 Jun;16(6):3141-3162. doi: 10.1038/s41596-021-00546-w. Epub 2021 Apr 30.
The global pandemic of coronavirus disease 2019 (COVID-19) highlights the shortcomings of the current testing paradigm for viral disease diagnostics. Here, we report a stepwise protocol for an RNA-extraction-free nano-amplified colorimetric test for rapid and naked-eye molecular diagnosis of COVID-19. The test employs a unique dual-prong approach that integrates nucleic acid (NA) amplification and plasmonic sensing for point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with a sample-to-assay response time of <1 h. The RNA-extraction-free nano-amplified colorimetric test utilizes plasmonic gold nanoparticles capped with antisense oligonucleotides (ASOs) as a colorimetric reporter to detect the amplified nucleic acid from the COVID-19 causative virus, SARS-CoV-2. The ASOs are specific for the SARS-CoV-2 N-gene, and binding of the ASOs to their target sequence results in the aggregation of the plasmonic gold nanoparticles. This highly specific agglomeration step leads to a change in the plasmonic response of the nanoparticles. Furthermore, when tested using clinical samples, the accuracy, sensitivity and specificity of the test were found to be >98.4%, >96.6% and 100%, respectively, with a detection limit of 10 copies/μL. The test can easily be adapted to diagnose other viral infections with a simple modification of the ASOs and primer sequences. It also provides a low-cost, rapid approach requiring minimal instrumentation that can be used as a screening tool for the diagnosis of COVID-19 at point-of-care settings in resource-poor situations. The colorimetric readout of the test can even be monitored using a handheld optical reader to obtain a quantitative response. Therefore, we anticipate that this protocol will be widely useful for the development of biosensors for the molecular diagnostics of COVID-19 and other infectious diseases.
2019 年冠状病毒病（COVID-19）的全球大流行凸显了当前病毒病诊断检测模式的不足。在这里，我们报告了一种逐步的方案，用于快速和肉眼分子诊断 COVID-19 的无 RNA 提取纳米扩增比色试验。该试验采用独特的双叉方法，将核酸（NA）扩增和等离子体传感集成在一起，用于即时检测严重急性呼吸系统综合征冠状病毒 2（SARS-CoV-2），样本检测时间小于 1 小时。无 RNA 提取纳米扩增比色试验利用带有反义寡核苷酸（ASO）的等离子体金纳米粒子作为比色报告物，检测 COVID-19 致病病毒 SARS-CoV-2 的扩增核酸。ASO 是 SARS-CoV-2 N 基因的特异性，ASO 与目标序列结合导致等离子体金纳米粒子聚集。这种高度特异性的聚集步骤导致纳米粒子的等离子体响应发生变化。此外，当使用临床样本进行测试时，该测试的准确性、灵敏度和特异性分别为>98.4%、>96.6%和 100%，检测限为 10 拷贝/μL。通过简单修改 ASO 和引物序列，该测试可以很容易地适应诊断其他病毒感染。它还提供了一种低成本、快速的方法，需要最小的仪器，可以作为资源匮乏情况下即时护理点 COVID-19 诊断的筛选工具。该测试的比色读数甚至可以使用手持光学读取器进行监测，以获得定量响应。因此，我们预计该方案将广泛用于 COVID-19 和其他传染病的分子诊断生物传感器的开发。
