High-surety isothermal amplification and detection of SARS-CoV-2, including with crude enzymes

Sanchita Bhadra; Timothy E Riedel; Simren Lakhotia; Nicholas D Tran; Andrew D Ellington

Given the low limits of detection we have observed, it is possible that LAMP-OSD might be used as part of diagnostics pipelines, or in direct patient screening. However, for this the reactions would need to operate under conditions commensurate with sample collection, especially in resource poor settings. Collection of nasopharyngeal and oropharyngeal swab specimens causes considerable discomfort to patients and requires supplies in the form of sterile swabs and transport media. Moreover, these samples are relatively difficult to self-collect. In contrast, saliva can be non-invasively collected simply by spitting in a sterile collection vessel and it can be done just as easily in a clinic as well as at home. Moreover, studies have shown that SARS-CoV-2 can be consistently detected in patient saliva with median viral loads of 3.3 × 10 6 copies/mL. 25 We tested the direct sample analysis ability of individual and multiplex LAMP-OSD assays by seeding them with 3 µL of human saliva spiked with SARS-CoV-2 virions. As control, duplicate LAMP-OSD reactions were seeded with virions suspended in 3 µL of TE buffer. Following 90 min incubation at 65 °C, endpoint observation of presence or absence of OSD fluorescence revealed that all assays seeded with SARS-CoV-2 virions, whether in human saliva or TE buffer, were brightly fluorescent (Figure 6 ). In contrast, assays lacking specific templates remained dark. These results suggest that LAMP-OSD assays might be used for direct analysis of human saliva samples in order to amplify and detect genetic signatures from SARS-CoV-2 virions.