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 Brian A. Rabea and Constance Cepkoa,b,c,1 aDepartment of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115; bHoward Hughes Medical Institute, Blavatnik Institute, Harvard Medical School, Boston, MA 02115 and cDepartment of Ophthalmology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115 Contributed by Constance Cepko, July 8, 2020 (sent for review June 2, 2020 reviewed by Roberto Bonasio and Michael Rosbash) The current severe acute respiratory syndrome coronavirus 2 pH indicator dye to visibly change color, is the optimal method for (SARS-CoV-2) pandemic has had an enormous impact on society a point-of-care LAMP-based diagnostic. worldwide, threatening the lives and livelihoods of many. The effects We designed and tested 11 sets of primers for the RT-LAMP will continue to grow and worsen if economies begin to open with- assay and used the LAMP reaction reagents from New England out the proper precautions, including expanded diagnostic capabil- Biolabs (NEB) for a colorimetric readout. These were tested ities. To address this need for increased testing, we have developed relative to other primers recently published by NEB. An optimal a sensitive reverse-transcription loop-mediated isothermal amplifica- set of primers directed toward a nonconserved region of the tion (RT-LAMP) assay compatible with current reagents, which utilizes SARS-CoV-2 Orf1a gene was identified as being particularly a colorimetric readout in as little as 30 min. A rapid inactivation pro- sensitive without being prone to background signals. In addition to tocol capable of inactivating virions, as well as endogenous nucleases, developing a robust RT-LAMP primer set, we also sought to was optimized to increase sensitivity and sample stability.
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