Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115.
Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115;
Proc Natl Acad Sci U S A. 2020 Sep 29;117(39):24450-24458. doi: 10.1073/pnas.2011221117. Epub 2020 Sep 8.
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.
当前严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）大流行已对全球社会造成巨大影响，威胁着许多人的生命和生计。如果经济在没有适当预防措施的情况下开始开放，包括扩大诊断能力，这些影响将继续增长和恶化。为了满足这一增加检测的需求，我们开发了一种与现有试剂兼容的敏感逆转录环介导等温扩增（RT-LAMP）检测方法，该方法可在 30 分钟内进行比色读数。优化了一种快速失活方案，该方案能够使病毒粒子以及内源性核酸酶失活，以提高灵敏度和样品稳定性。该方案与 RT-LAMP 检测方法相结合，在每个微升样本中至少有 50 个病毒 RNA 拷贝的灵敏度。为了进一步提高灵敏度，开发了一种与这种失活方法兼容的纯化方案。失活和纯化方案与 RT-LAMP 检测方法相结合，使灵敏度提高到每个微升样本中至少有 1 个病毒 RNA 拷贝。这种简单的失活和纯化方案成本低廉，与其他下游 RNA 检测平台兼容，并且使用现成的试剂。它应该会增加 SARS-CoV-2 检测的可用性，并扩大可以进行这种检测的环境。
