Martin Coleman D, Bender Andrew T, Sullivan Benjamin P, Lillis Lorraine, Boyle David S, Posner Jonathan D
Department of Chemical Engineering, University of Washington Seattle Washington USA.
Department of Mechanical Engineering, University of Washington Seattle Washington USA.
Sens Diagn. 2024 Jan 12;3(3):421-430. doi: 10.1039/d3sd00246b. eCollection 2024 Mar 14.
Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.
用于检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的核酸扩增检测已成为2019冠状病毒病（COVID-19）大流行期间的一种重要检测机制。虽然这些传统的核酸诊断方法具有高度的敏感性和选择性，但它们不适合家庭或门诊使用。相比之下，快速抗原检测具有成本效益且用户友好，但缺乏敏感性和特异性。在此，我们报告了一种以MS2噬菌体作为全流程对照的单管、双重逆转录酶重组酶聚合酶扩增SARS-CoV-2检测方法的开发。检测通过实时荧光或侧向流读数进行，分析灵敏度为每个反应50个拷贝。与先前发表的检测方法不同，基于RNA的MS2噬菌体对照报告了裂解、逆转录和扩增的成功操作。这种SARS-CoV-2检测方法具有高灵敏度检测、通过侧向流免疫层析试纸条进行可视化读数、在不到25分钟内得出结果、所需仪器最少以及具有有用的流程内部控制以排除假阴性检测结果等特点。
