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测试范式的不足。在这里，我们报告了一种用于 COVID-19 快速和裸眼分子诊断的无 RNA 提取纳米放大比色测试的逐步协议。该测试采用独特的双管齐下方法，将核酸 (NA) 扩增和等离子体传感相结合，用于即时检测严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2)，并具有样本到检测反应<1小时的时间。无 RNA 提取的纳米放大比色测试利用反义寡核苷酸 (ASO) 覆盖的等离子体金纳米粒子作为比色报告基因，检测来自 COVID-19 致病病毒 SARS-CoV-2 的扩增核酸。ASO 对 SARS-CoV-2 N 基因具有特异性，并且 ASO 与其目标序列的结合导致等离子体金纳米粒子的聚集。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，发现该测试的准确度、灵敏度和特异性分别为 >98.4%、>96.6% 和 100%，检测限为 10 拷贝/μL。通过对 ASO 和引物序列进行简单修改，该测试可以很容易地适用于诊断其他病毒感染。它还提供了一种低成本、快速的方法，需要最少的仪器，可以用作在资源匮乏情况下的即时护理环境中诊断 COVID-19 的筛查工具。甚至可以使用手持式光学阅读器监测测试的比色读数以获得定量响应。因此，我们预计该协议将广泛用于开发用于 COVID-19 和其他传染病分子诊断的生物传感器。