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

In some experiments, LAMP-OSD reactions were assembled using Bst-LF instead of Bst 2.0 and RTX. This was done by replacing Bst 2.0 and RTX in the LAMP-OSD reactions with ~2 x 10 7 CFU of rehydrated Bst-LF cellular reagents. 20 These cellular reagents were prepared in an E. coli BL21(DE3) derivative ∆endA ∆recA strain using previously described protocols. 20 Briefly, chemically competent bacteria were transformed with Bst-LF expression plasmid. Overnight 3 ml cultures of these transformed bacteria were grown in 2X YT broth containing 100 µg/mL ampicillin. Subsequently, sub-cultures were initiated at 1:200 dilution using 50 mL Superior Broth TM (Athena Environmental Sciences, Inc., Baltimore, MD, USA) containing 100 µg/ml ampicillin. These cultures were incubated at 37 °C and constant 225 rpm agitation till they reached the log phase of growth (A600 = 0.4 to 0.7). Protein production was initiated by adding 20ng/ml anhydrotetracycline (aTC) followed by 3 h incubation at 37 °C with 225 rpm agitation. After induction, bacteria were collected by centrifugation followed by washing twice in cold 1X PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4). The bacterial pellets were resuspended in cold 1X PBS at a density of A600 = 3.5 to 6.5. Some 2x10 8 induced bacteria (estimated from the A600 value using the relation 0.5 optical density = 5x10 8 bacteria/ml) were aliquoted into individual 0.2 ml PCR tubes and frozen at -80 °C overnight prior to lyophilization for 3 h at 197 mTorr and -108 °C using a VirTis Benchtop Pro lyophilizer (SP Scientific, Warminster, PA, USA). Immediately before use, each tube of cellular reagents was hydrated in 30 µL water and 3 µL of this suspension was added to each LAMP reaction.