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

We chose to design OSD probes for three of these pre-published LAMP primer sets, from here on referred to as the Tholoth, Lamb, and NB primers. The three primer sets target different regions in the ORF1AB and N genes of the SARS-CoV-2 genome. Fluorogenic OSD probes were designed for each of these primer sets using previously described principles and the Nucleic acid circuit design software NUPACK available freely at http://www.nupack.org/. 10, 19 Briefly, the target derived loop regions between the F1c and F2 primer binding sites were chosen as OSD binding regions for each of the three LAMP primer sets. The long OSD strand was designed to be complementary to this loop region. Single stranded 11-12 nucleotides long toehold regions were designated on one end of this long strand while a complementary short OSD strand was designed to hybridize to the remaining portion of the long strand. The long strand was labeled with a fluorescein moiety at the terminus not acting as the toehold. The short strand was labeled with a quencher and all free 3'-OH ends were blocked with inverted dT to prevent extension by DNA polymerase. Amplicon accumulation was measured by adding OSD probes. First, Tholoth, Lamb, and NB OSD probes were prepared by annealing 1 µM of the fluorophore-labeled OSD strand with 2 µM, 3 µM, and 5 µM, respectively of the quencher-labeled strand in 1X Isothermal buffer. Annealing was performed by denaturing the oligonucleotide mix at 95 °C for 1 min followed by slow cooling at the rate of 0.1 °C/s to 25 °C. Excess annealed probes were stored at -20 °C. Annealed Tholoth, Lamb, and NB OSD probes were added to their respective LAMP reactions at a final concentration of 100 nM, 100 nM, and 20 nM, respectively of the fluorophore-bearing strand. Multiplex LAMP-OSD assays comprising both Tholoth and NB primers and probes were set up using the same conditions as above except, the total LAMP primer amounts were made up of equimolar amounts of Tholoth and NB primers.