Reducing polymerase-related cost

[Kevin Mahaffey]

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?

- Saliva seems to carry sufficient viral load: To, et. al. found that saliva samples were acceptable for SARS-CoV-2 RT-qPCR assays from hospitalized patients

- 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!

-Kevin

[Jonathan Leo Schmid-Burgk]

Hi Kevin,

Thanks for your great suggestions!

- We have not titrated down Bst 3.0 yet, but agree very much this could be the easiest way to bring down cost.

- Reducing RT-LAMP volume when using saliva could be a good option in some scenarios, albeit it would require reliable dosing as you pointed out. We will validate the swab-to-LAMP approach first using larger volumes to submerge the swab, but will follow closely what other groups are establishing in the colorimetric LAMP space - all these developments should be compatible with LAMP-Seq

Best,

Jonathan

[Jingwei Zhang]

I wonder with the specificity that I we may achieve (needed for cross-reactivity check anyway).

Could we use live E. coli rather than purified enzymes?

[Dineshkumar Dandekar]

Not possible to use live E.Coli as the sample RNA will be degraded due to RNAse from cells

[Mikolaj Slabicki]

Good point Dineshkumar.

[clemen...@gmail.com]

Hello,

I am just curious to know if you maybe tried your protocol using a homemade Bst, like the Bst v5.9 developed in 2018 by the Ellington lab?

If not, would you say it is doable? (It is difficult to find much details about Bst 3.0 to compare it with)

best

Clément

[Mikolaj Slabicki]

Hi Clement.

Thanks for your suggestion. Yes, it might be worth testing Bst v5.9.

Best,

Mikolaj

[peter....@worldmosquito.org]

Hi all, 

-We regularly use colorimetric LAMP for wolbachia diagnostics and have found that by reducing the reaction volume down to 17uL, we are still able to generate a comparable LOD. 

I would suspect that you would be able to titrate this further in this use case. 

-We've experimented using sous vide devices in the past and were able to generate concordant results across multiple runs.. You can do it - but there's a big practicality question, in a low resource setting - yes possibly.  Saying that, we've found in most of the sites that we work that heat blocks are more readily available than sous vide devices.

Cheers,

Peter

[Mikolaj Slabicki]

Hi Peter,

Thanks a lot for the suggestion. I think the reduction of the LAMP volumes might be challenging for swabs, but probably feasible for saliva. 

Minimal reaction volume will be potentially useful in the later phases of the protocol optimization.

Best,

Mikolaj

[daniel...@gmail.com]

Hi Mikolaj,

Are you evaluating the protocol using saliva as well?

That's the final frontier in this being an absolute game-changer in mass testing, solving the various logistical bottlenecks.

Really great round-up on Genome Web:

https://www.genomeweb.com/molecular-diagnostics/covid-19-testing-could-get-boost-alternative-sample-types

Let me just add that Illumina is excellent at processing saliva samples and could help with offering some guidance to lab operations.

[bio...@gmail.com]

Regarding saliva matrix.  I'm looking forward to someone assessing sensitivity of samples used with mucolytics: https://testingmethods.crowdicity.com/post/3193060

Cheers,

AJP

[bio...@gmail.com]

Would be possible if we have an E. coli with no RNase activity.

[Jingwei Zhang]

Same thoughts, shake hands.

Do you have any academic strain in mind?

NEB could have a lot of industrial strains with partial nuclease KOs.