Hi there—fantastic work to the team validating the protocol.
Apologies if you've already covered these in discussion, but wanted to ask a few questions into reducing per-sample polymerase cost potentially 100-fold (64 U/mL @ 25 uL reaction volume)?
Reducing Bst 3.0 concentration below 320 U/mL
- 64 U/mL [or lower] may be acceptable within incubation time target: Lucigen has some data
in their optimization materials indicating results at 20% standard concentration (time-to-positive increasing from ~10 min to ~20 min) and no meaningful increase in non-template amplification—unsure if this is internal data; I couldn't find the references
Reducing reaction volume and need for a swab (supply-chain limited, cost driver): Is the main requirement for a large reaction volume (500 uL vs. 25 uL typical for a LAMP reaction) handling crude samples from a swab? If so, can small but variable (5-20 uL) saliva samples suffice to get us to a ~25 uL nominal reaction volume?
- 20% of typical primer concentrations may be acceptable: The same Lucigen data
above indicates acceptable performance at as low as 20% of normal primer volume. Coupled with enzyme concentration variability above, that seems to indicate tolerance for large fluctuations in sample volume.
- Does the saliva sample matrix have inhibitory effects at high concentration? I am not aware of any research covering this.
- Can a non-standard and widely available collection device (e.g. small paper/plastic strip, toothpick, small plastic straw) reliably collect low-volume saliva within acceptable bounds? I'm not aware of any research here, though Tsai, et. al. were able to reliably detect STATH expression
in saliva collected from cigarette butts which may approximate what we're talking about here.
Finally, one other cost optimization may be to use commercially available sous vide devices (<$200, +/- 0.1 C variability) for LAMP incubation.
Again—thank you all for the hard work on this!