RT-LAMP Detection of SARS-CoV-2 in Swabs and Saliva

The RT-LAMP primer sets used in this study were designed by Lamb et al. (2020) [20 (link)] against the nonstructural protein 3 (NSP3) coding region of ORF1ab. The RT-LAMP reaction for swab and saliva was conducted in a total volume of 25 μL of 1X isothermal amplification buffer, 1.4 mM deoxynucleoside triphosphates (dNTPs), 6 mM MgSO4, 1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM Loop F/B primers, 0.32 U/µL Bst 2 WarmStart DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA), 15 U/µL WarmStart RTx Reverse Transcriptase (New England Biolabs), 8 µL of nuclease-free water, and 2 µL of viral RNA. A no-template control (NTC) with the same water used for the reaction contained substituted viral RNA. The reaction mixtures were incubated using a PCR thermocycler at 63 °C for 45 min for the swab samples and at 65 °C for 60 min for the saliva samples followed by enzyme inactivation at 80 °C for 10 min. Amplification products were fractionated by 3% agarose gel electrophoresis, stained with fluorescent dye ethidium bromide, and visualized under ultraviolet (UV) light.