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Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary patient samples

By Dawn M Dudley, Christina M. Newman, Andrea M Weiler, Mitchell D. Ramuta, Ceclia G. Shortreed, Anna S. Heffron, Molly A Accola, William M Rehrauer, Thomas C Friedrich, David H O'Connor

Posted 02 Sep 2020
medRxiv DOI: 10.1101/2020.08.30.20184796

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR and we provide a revised method to work directly with saliva as starting material. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

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