Rapid isothermal amplification and portable detection system for SARS-CoV-2.
Rapid isothermal amplification and portable detection system for SARS-CoV-2.
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DOI：
10.1073/pnas.2014739117
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发表时间：
2020-09-15
影响因子：
11.1
通讯作者：
Bashir R
中科院分区：
文献类型：
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作者：
Ganguli A;Mostafa A;Berger J;Aydin MY;Sun F;Ramirez SAS;Valera E;Cunningham BT;King WP;Bashir R
目前用于检测SARS-CoV-2的检测方法的一个重要局限性源于它们依赖于耗时、劳动密集型和基于实验室的病毒分离、裂解和去除抑制物质的方案。虽然RT-PCR仍然是进行临床诊断以扩增RNA序列的金标准，但迫切需要快速、准确、简单和便携的替代测试平台。在这里，我们展示了SARS-CoV-2的等温RT-LAMP核酸检测，使用增材制造的检测盒和基于智能手机的仪器进行检测，可以在样本采集点进行。COVID-19疫情提供了一个紧迫的例子，说明最先进的诊断方法的可用性与当前需求之间存在差距。随着测定方案和引物序列变得广为人知，许多实验室使用诸如RT-PCR或逆转录环介导等温扩增（RT-LAMP）的方法进行诊断测试。在这里，我们报告了一种RT-LAMP等温检测严重急性呼吸综合征冠状病毒2（SARS-CoV-2）病毒，并证明了临床样本的检测使用一个简单和方便的床旁（POC）仪器。我们通过将拭子浸入加标病毒的合成鼻液中，将拭子移至病毒转运培养基（VTM）中，并在无RNA提取试剂盒的情况下对一定体积的VTM进行取样以进行RT-LAMP测定来表征该测定。该检测方法的检测限（LOD）为50 RNA拷贝/μL的VTM溶液在30分钟内。我们进一步证明我们的检测方法检测SARS-CoV-2病毒从20个临床样本。最后，我们展示了一种便携式和实时POC设备，使用增材制造的三维盒和基于智能手机的读取器从VTM样品中检测SARS-CoV-2。POC系统进行了测试，使用10个临床样本，并能够检测SARS-CoV-2从这些临床样本区分阳性样品和阴性样品后30分钟。POC测试是完全一致的RT-PCR控制。这项工作证明了SARS-CoV-2诊断的替代途径，不需要传统的实验室基础设施，在样本采集点需要诊断的环境中。
An important limitation of current assays for the detection of SARS-CoV-2 stems from their reliance on time-consuming, labor-intensive, and laboratory-based protocols for viral isolation, lysis, and removal of inhibiting materials. While RT-PCR remains the gold standard for performing clinical diagnostics to amplify the RNA sequences, there is an urgent need for alternative testing platforms that are rapid, accurate, simple, and portable. Here, we demonstrate isothermal RT-LAMP nucleic acid-based detection of SARS-CoV-2 with an additively manufactured cartridge and a smartphone-based instrument for testing that can be performed at the point of sample collection. The COVID-19 pandemic provides an urgent example where a gap exists between availability of state-of-the-art diagnostics and current needs. As assay protocols and primer sequences become widely known, many laboratories perform diagnostic tests using methods such as RT-PCR or reverse transcription loop mediated isothermal amplification (RT-LAMP). Here, we report an RT-LAMP isothermal assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and demonstrate the assay on clinical samples using a simple and accessible point-of-care (POC) instrument. We characterized the assay by dipping swabs into synthetic nasal fluid spiked with the virus, moving the swab to viral transport medium (VTM), and sampling a volume of the VTM to perform the RT-LAMP assay without an RNA extraction kit. The assay has a limit of detection (LOD) of 50 RNA copies per μL in the VTM solution within 30 min. We further demonstrate our assay by detecting SARS-CoV-2 viruses from 20 clinical samples. Finally, we demonstrate a portable and real-time POC device to detect SARS-CoV-2 from VTM samples using an additively manufactured three-dimensional cartridge and a smartphone-based reader. The POC system was tested using 10 clinical samples, and was able to detect SARS-CoV-2 from these clinical samples by distinguishing positive samples from negative samples after 30 min. The POC tests are in complete agreement with RT-PCR controls. This work demonstrates an alternative pathway for SARS-CoV-2 diagnostics that does not require conventional laboratory infrastructure, in settings where diagnosis is required at the point of sample collection.