SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.
SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.
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DOI：
10.1073/pnas.2011221117
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发表时间：
2020-09-29
影响因子：
11.1
通讯作者：
Cepko C
中科院分区：
文献类型：
--
作者：
Rabe BA;Cepko C
这项工作描述了SARS-CoV-2诊断测试的样品制备和检测管道的优化，该测试快速且不需要专门的设备。该管道包括病毒灭活，使样品更安全地工作，然后是灵敏的30分钟等温检测反应，基于颜色的红色到黄色读数。灵敏度可以进一步提高使用简单和廉价的纯化方案。该管道可以帮助解决测试容量短缺的问题，并且可以在各种环境中运行。目前的严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）大流行对全世界社会产生了巨大影响，威胁到许多人的生命和生计。如果经济开始开放而没有适当的预防措施，包括扩大诊断能力，这种影响将继续增长和恶化。为了解决这一需要增加测试，我们已经开发了一个敏感的逆转录环介导的等温扩增（RT-LAMP）检测兼容目前的试剂，它利用比色读数在短短30分钟。一个快速灭活方案能够灭活病毒粒子，以及内源性核酸酶，进行了优化，以提高灵敏度和样品的稳定性。该方案与RT-LAMP测定相结合，具有样品中至少50个病毒RNA拷贝/微升的灵敏度。为了进一步提高灵敏度，开发了与该灭活方法兼容的纯化方案。灭活和纯化方案与RT-LAMP测定相结合，使样品中的灵敏度达到每微升至少1个病毒RNA拷贝。这种简单的灭活和纯化管道价格低廉，与其他下游RNA检测平台兼容，并使用容易获得的试剂。它应该增加SARS-CoV-2测试的可用性，并扩大可以进行这种测试的设置。
This work describes the optimization of a sample preparation and detection pipeline for a SARS-CoV-2 diagnostic test that is rapid and does not require specialized equipment. This pipeline consists of viral inactivation, rendering samples safer to work with, followed by a sensitive 30-min isothermal detection reaction with a color-based red to yellow readout. Sensitivity can be further improved using a simple and inexpensive purification protocol. This pipeline can help address the shortage of testing capacity and can be run in a variety of settings. The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.
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DOI：
10.1126/science.aas8836
发表时间：
2018-04-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Myhrvold C;Freije CA;Gootenberg JS;Abudayyeh OO;Metsky HC;Durbin AF;Kellner MJ;Tan AL;Paul LM;Parham LA;Garcia KF;Barnes KG;Chak B;Mondini A;Nogueira ML;Isern S;Michael SF;Lorenzana I;Yozwiak NL;MacInnis BL;Bosch I;Gehrke L;Zhang F;Sabeti PC
通讯作者：
Sabeti PC
影响因子：
3.7
作者：
Poole CB;Li Z;Alhassan A;Guelig D;Diesburg S;Tanner NA;Zhang Y;Evans TC Jr;LaBarre P;Wanji S;Burton RA;Carlow CK
通讯作者：
Carlow CK
影响因子：
4
作者：
Chen, Jingming;Chin, Adam;Taboas, Juan M.
通讯作者：
Taboas, Juan M.
影响因子：
4.4
作者：
DEJONG, MH;VANDERHOEVEN, JS;OLIJVE, JH
通讯作者：
OLIJVE, JH
影响因子：
3.3
作者：
Nagamine, K;Hase, T;Notomi, T
通讯作者：
Notomi, T