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

Loop-mediated isothermal amplification (LAMP) uses the strand-displacing Bst DNA polymerase and 4 primers (FIP, BIP, F3, and B3) that bind to 6 target regions (B3, B2, B1, F1c, F2c and F3c) to generate 10 9 to 10 10 copies of DNA or RNA targets, typically within 1 to 2 h (Figure 1 ). 1 In greater detail, F2 in FIP (F1c-F2) and B2 in BIP (B1c-B2) initiate amplification. F1c and B1c self-prime subsequent amplification. F3 and B3-initiated DNA synthesis displaces FIP and BIP-initiated strands. 3′-ends of the resulting single-stranded, dumbbell-shaped amplicons are extended to hairpins by Bst polymerase. FIP and BIP hybridize to the single-stranded loops and initiate DNA synthesis that opens the hairpin to form concatameric amplicons containing self-priming 3′-end hairpins. The ensuing continuous amplification generates double-stranded concatameric amplicons with self-priming hairpins and single-stranded loops. 1 LAMP can rival PCR for sensitivity without thermocycling, 2 and additional stem and loop primers can accelerate amplification, with some LAMP assays being complete within 10 min. 3, 4 However, since LAMP is commonly read using non-specific methods (such as, Mg 2+ precipitation, intercalating dyes or labeled primers) that cannot distinguish spurious amplicons that frequently arise from continuous amplification, its utility can be limited. We have previously overcome these drawbacks using oligonucleotide strand exchange (OSD) probes, based in part on advances in strand exchange DNA computation (Figure 1) . Strand exchange occurs when two partially or fully complementary strands hybridize to each other by displacing pre-hybridized strand(s) ( Figure 1B) . Strand exchange usually initiates by basepairing at single-stranded 'toeholds' and progresses to form additional basepairs via branch migration, allowing the rational design of complex algorithms and programmable nanostructures [5] [6] [7] [8] [9] . The hemiduplex oligonucleotide probes contain a so-called 'toehold' that allows sequence-specific interaction with a target molecule, and have opposed fluor and quencher moieties. In the presence of a complementary target, the OSD probes can undergo strand exchange and separation, leading to an easily read fluorescent signal. 10 In essence the OSD probes are functional equivalents of TaqMan probes and have been shown to accurately report single or multiplex LAMP amplicons from few tens of targets without interference from non-specific amplicons or inhibitors. 10, 11 Of equal import, the programmability of OSD probes allows their adaptation to many different assay formats, including to off-the-shelf devices such as glucometers and pregnancy test strips. [12] [13] [14] [15] [16] Figure 1. LAMP-OSD schematic. 'c' denotes complementary sequences. F and Q on the OSD denote fluorophore and quencher, respectively. OSD and subsequent strand exchange intermediates are denoted by numbered domains, which represent short (usually <12 nt) sequences in an otherwise continuous oligonucleotide. Complementary domains are indicated by asterisk.