Abstract：
Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.
摘要：
必须进行高度敏感和具体的现场测试，以加强对未来新出现的传染病的准备。这里,我们描述了能够将特定DNA序列的识别转化为放大的比色信号的Cas12a介导的DNAzyme致动器的构建。为了解决现场应用的病毒RNA提取挑战，我们开发了一种快速有效的方法，能够裂解病毒颗粒，保存释放的病毒RNA，浓缩病毒RNA。将DNAzyme致动器与病毒RNA提取方法和环介导的等温扩增相结合，可以进行流线型比色测定，以高灵敏度比色检测漱口和唾液中的呼吸道RNA病毒。该测定可以检测少至83个含漱液中的病毒颗粒/100μL和166个唾液中的病毒颗粒/100μL。整个化验,从样品处理到视觉检测，在单个受控温度下在1小时内完成。我们通过在207个含漱液和唾液样本中检测SARS-CoV-2来验证该测定法，达到96.3%的临床灵敏度和100%的特异性。该测定适用于检测其他病原体中的特定核酸序列,并且适用于资源有限的环境。
