由严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 引起的 2019 年冠状病毒病 (COVID-19) 已引起全球大流行,凸显了对快速和便携式诊断方法的空前需求。基于成簇规律间隔的短回文重复序列 (CRISPR)/CRISPR 相关 (Cas) 蛋白的平台已用于检测病原体。然而,在进一步的应用和研究中,由于需要多个步骤,许多方法显示交叉反应的风险增加。热稳定的 Cas12b 可将等温扩增和 CRISPR 介导的检测相结合,从而降低交叉污染的风险。在这项研究中,我们开发了一种便携式特异性诊断方法,将金纳米粒子 (AuNP) 与热稳定的 CRISPR/Cas12b 增强逆转录环介导等温扩增 (RT-LAMP) 相结合,称为 SCAN,以区分 SARS-N 基因来自流感基因的 CoV-2。我们使用来自质粒转染细胞的 RNA 验证了我们的方法。基于 AuNP 的强摩尔吸收系数,我们可以通过肉眼轻松区分阳性结果。此外,由于操作方便,SCAN 具有进行高通量测试的潜力。综上所述,SCAN打破了传统检测方法的场地和设备限制,可以应用在医院和临床实验室之外,大大扩展了COVID-19的检测范围。以区分 SARS-CoV-2 的 N 基因和流感基因。我们使用来自质粒转染细胞的 RNA 验证了我们的方法。基于 AuNP 的强摩尔吸收系数,我们可以通过肉眼轻松区分阳性结果。此外,由于操作方便,SCAN 具有进行高通量测试的潜力。综上所述,SCAN打破了传统检测方法的场地和设备限制,可以应用在医院和临床实验室之外,大大扩展了COVID-19的检测范围。以区分 SARS-CoV-2 的 N 基因和流感基因。我们使用来自质粒转染细胞的 RNA 验证了我们的方法。基于 AuNP 的强摩尔吸收系数,我们可以通过肉眼轻松区分阳性结果。此外,由于操作方便,SCAN 具有进行高通量测试的潜力。综上所述,SCAN打破了传统检测方法的场地和设备限制,可以应用在医院和临床实验室之外,大大扩展了COVID-19的检测范围。由于操作方便,SCAN 具有进行高通量测试的潜力。综上所述,SCAN打破了传统检测方法的场地和设备限制,可以应用在医院和临床实验室之外,大大扩展了COVID-19的检测范围。由于操作方便,SCAN 具有进行高通量测试的潜力。综上所述,SCAN打破了传统检测方法的场地和设备限制,可以应用在医院和临床实验室之外,大大扩展了COVID-19的检测范围。
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Visual detection of SARS-CoV-2 with a CRISPR/Cas12b-based platform
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19.