The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5′-6FAM 3′-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

SARS-CoV-2引起的COVID-19传染病暴发严重威胁着人类的生命。迫切需要一种快速，可靠和特定的检测方法。在本文中，我们报道了采用LAMP和CRISPR / Cas12a技术的SARS-CoV-2无污染视觉检测方法。CRISPR / Cas12a试剂已预先添加到管盖的内壁上。LAMP反应后，将CRISPR / Cas12a试剂流入试管中，并用手摇晃使其与扩增子溶液混合，可有效避免扩增后重新打开盖子可能造成的扩增子形成的气溶胶污染。CRISPR / Cas12a可以高度特异性地识别靶序列，并区分切割单链DNA探针（5'-6FAM 3'-BHQ1）。借助智能手机和便携式3D打印仪器，无需任何专用仪器就可以用肉眼看到产生的荧光，这在即时检测中很有希望。整个扩增和检测过程可在40分钟内完成，并具有20个拷贝的SARS-CoV-2 RNA的高灵敏度。该反应具有高特异性，可以避免与其他常见病毒（如流感病毒）的交叉反应。对于浙江省疾病预防控制中心提供的7例阳性和3例阴性的拭子样本，我们的检测结果具有100％的阳性一致性和100％的阴性一致性，证明了该方法的准确性和应用前景。整个扩增和检测过程可在40分钟内完成，并具有20个拷贝的SARS-CoV-2 RNA的高灵敏度。该反应具有高特异性，可以避免与其他常见病毒（如流感病毒）的交叉反应。对于浙江省疾病预防控制中心提供的7例阳性和3例阴性的拭子样本，我们的检测结果具有100％的阳性一致性和100％的阴性一致性，证明了该方法的准确性和应用前景。整个扩增和检测过程可在40分钟内完成，并具有20个拷贝的SARS-CoV-2 RNA的高灵敏度。该反应具有高特异性，可以避免与其他常见病毒（如流感病毒）的交叉反应。对于浙江省疾病预防控制中心提供的7例阳性和3例阴性的拭子样本，我们的检测结果具有100％的阳性一致性和100％的阴性一致性，证明了该方法的准确性和应用前景。