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

Although there are several DNA polymerases (such as Bsu, Phi29, Klenow, Vent, and Deep Vent) that can catalyze strand displacement DNA synthesis, Bst DNA polymerase has been reported to be the most efficient at performing LAMP. 1 For the detection of RNA targets, reverse transcription LAMP commonly uses a combination of Bst and a dedicated reverse transcriptase, such as AMV reverse transcriptase. That said, it is known that some targets and primers are amenable to reverse transcription LAMP using only the Bst enzyme. The Bst DNA polymerase large fragment . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.039941 doi: bioRxiv preprint (Bst-LF; the precursor to the commercial version Bst 2.0 from New England Biolabs) possesses innate reverse transcription ability and can be used for LAMP. 1, 26 Moreover, Bst-LF can support LAMP even when used in the form of cellular reagents, dried bacteria containing overexpressed enzymes that can be added directly to reactions (see also below). 20 Considering potential supply chain issues, especially for resource poor settings, we sought to determine whether Bst-LF (an open source enzyme that maybe produced on site, see also attached protocol for production, Supplementary information) as a cellular reagent could be used for LAMP-OSD assays with SARS-CoV-2 targets. The previously described NB, Tholoth, and 6 primer Lamb LAMP-OSD assays were carried out with SARS-CoV-2 viral genomic RNA and Bst-LF enzyme as a cellular reagent 20 in an E. coli BL21(DE3) derivative ∆endA ∆recA strain (see also attached protocol for production, Methods).