Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples
Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples
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DOI：
10.1021/acs.analchem.0c05170
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发表时间：
2021-05-25
影响因子：
7.4
通讯作者：
Bashir, Rashid
中科院分区：
文献类型：
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作者：
Ganguli, Anurup;Mostafa, Ariana;Bashir, Rashid
COVID-19疫情凸显了部署最先进诊断平台的缺点。虽然几种基于聚合酶链反应（PCR）的技术已经迅速发展，以满足日益增长的测试需求，这些技术往往需要通过拭子收集的样本，使用RNA提取试剂盒，和昂贵的热循环仪，以成功地进行测试。最近还证明了基于等温扩增的方法可用于快速检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2），方法是通过最大限度地减少样品制备，同时降低仪器和反应复杂性。此外，唾液作为样本来源的报告有限，其中一些报告表明，当比较逆转录环介导等温扩增（RT-LAMP）与基于PCR的技术时，灵敏度较差。在本文中，我们证明了一种改进的灵敏度测定从唾液中使用两步RT-LAMP测定，其中一个简短的10分钟RT步骤进行，只有B3和反向内部引物在最终反应之前。我们表明，虽然一步RT-LAMP表现出令人满意的结果，优化的两步方法允许每个反应只检测几个分子，并执行显着优于一步RT-LAMP和传统的两步RT-LAMP方法与RT步骤中包括的所有引物。我们展示了RT-PCR的对照测量，重要的是，我们展示了用于从病毒转运介质和唾液临床样本中检测SARS-CoV-2的基于RNA提取的RT-LAMP检测。
The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.