[Alejandro Caro-Quintero]

Dear all,

thank you so much for providing this open protocols for everybody, I am currently involved in SARS-CoV2 detection in Colombia, and we are currently trying to start setting up a commercial kit vs the BOMB.bio protocol, we have some questions about the preparation of the GITC buffer, specifically what options in terms of reagents we have for antifoam.

Have you try anything in your protocols as the following video (https://www.youtube.com/watch?v=pdqjFHkTjEQ). Do you think this will improve speed?, what could be the risks of doing such protocols?

The other thing I am wondering is if any of you know what are the requirements for protocol validation, I think that depends on the country but I would like to have some guidelines.

Finally, is there any open source for automating the extraction.

Thank,

Alejandro

[TimH]

Hi Alejandro – best of luck with your efforts. The anti-foam is an optional component as far as I am aware – it is the GITC and detergent that will lyse cells (and virus). So if you don’t have it, then you might want to still try without it.

We have not tried the V&P tool as in the video. I would think it is fine in principle, however, to avoid contamination you would need a new microplate and wash buffer for every plate. So might not actually save much on plastic ware. Robotic automation, including OpenTrons will work (likely more details/protocols will emerge around this soon); however, multichannel pipettes are also fast and effective if you don’t have one. The most time consuming aspect is usually getting the lysate from the swab in individual tubes into a 96-well format.

Best wishes,

TimH

[admin]

Ahoy,

Two things.

First I have actually tried using the BOMB 2x GITC Buffer without Antifoam. I did not test for actual efficiency as this was just a rushed little experiment were the focus was on testing a new enzyme down the line rather than the extraction itself. The reason I left out the anti-foam was simply that I was in a hurry, had to make fresh 2xGITC buffer and could not find the anti-foam right away. Anyhow at least on the gel the DNA extract looked great. However, pipetting the Buffer without anti-foam is an entirely different matter, it is a bit of a nightmare. But, as soon as the Isoprop gets even close, the foam instantaneously collapses. As I have also added the first three components (lysis buffer/ beads/isopropanol) in varying order without seeing any difference in performance. I can imagine that a possible solution would be to make a lysis buffer + isopropanol mastermix just before adding the two to your sample, followed by adding the beads.

Second, if it would make things easier for you to have someone in your own timezone who has used several of the BOMB protocols extensively, a member of the wider BOMB team is actually in Colombia. If you send us a message via the contact form we can connect you.

Hope any of this helps.

Cheers

Tim M

 

**edit**
I have talked to a colleague about my remark and he reminded me that the protocol I was using involved Proteinase-K lysis pre GITC/ISOp/Bead addition. Considering this, maybe it would be a better idea to just mix a fraction of the Isopropanol with the GITC buffer rather than combining all of the two components before addition. Using Isoprop.l as the Antifoam so to speak while still maintaining most of the GITC buffer concentration. An then after GITC buffer lysis the remaining Isoprop. could be added. Maybe someone more Biochemistry versed can chime in?

 

[Alejandro Caro-Quintero]

Dear All,

we have started diagnosis at my institution (AGROSAVIA, Colombia)  two weeks ago and we have been running in parallel both the column extraction kit and your protocol for around 20 samples.  We have noticed that the RNaseP gene amplifies at the same CT (human RNA extraction internal control), however, the RdRP gene (SARS-Cov2) amplifies around three CT after.  Another observation is that whenever there is Blood in the samples we failed to amplify the RdRP gene, beads seem to get attached to the blood. For these samples, we try a  1/4 dilution and got RdRP to amplify.

To improve the RNA extraction for the virus, we plan to increase 50% and 100% the volume of beads in the protocol.

We would like to know if you have any suggestions, for those samples with blood and to increase the extraction efficiency of viral RNA. Do you think a higher RNA extraction efficiency can be achieved by adding the RNA binding buffer (protocol 8.2 with Gu-HCl)?

PS. I know Oscar Javier Ortega, who was one of my students at a class at UNIANDES, and who has been of enormous help to start this protocol.

Best,

Alejandro

[Alexander James Phillips]

Hi Alejandro,

I don’t know if you got an answer to your question?:

higher RNA extraction efficiency can be achieved by adding the RNA binding buffer (protocol 8.2 with Gu-HCl)?

Regarding:

we plan to increase 50% and 100% the volume of beads in the protocol.

I know that TimH and TimM both suggested various approaches for increasing yields which from this comment https://bomb.bio/forums/topic/the-official-sars-cov-2-bomb-extraction-protocol#post-9318 and 1 above seemed like: more beads / high bead concentration, smaller bed diameter (higher surface area).

Separately, I’m looking into clinical relevant synthetic sample standards for SARS-CoV-2 and your comment about clots of blood making beads stick to it was very interesting.  If you had a photo or more info you could send that would be much appreciated.  Please feel free to contact me on bioajp at gmail dot com

Yours sincerely and with best wishes,
AJP

p.s. when you are using Bomb.Bio for clinical sample processing please send a message to let everyone know 🙂

[Alexander James Phillips]

Alejandro and colleagues published their work.  I did a rough translation to English here.  They add Proteinase K to samples with blood.  They also have a different protocol (dilute with saline) for samples that have more mucus.  These links can also be found in the spreadsheet of Bomb.bio adopters / validators.

[Rob Page]

Hi Alejandro,

I am working at King’s College London and Guy’s Hosptial to set up and validate as many RNA extraction protocols for SARS-CoV-2 as possible, including several silica based methods we have developed in house. We are seeing the same thing as you described. When compared to commercial kits, our RNaseP CTs are exactly the same, but we lose 2-3 CT on our CoV primer-probes (CDC N1 & N2).

Interestingly we do not see this effect if we dilute our samples 1:10 (although we lose sensitivity by all methods), so our working hypothesis is that gDNA (larger) may be outcompeting the viral RNA (smaller) for binding where there is a high overall concentration of nucleic acid. The RNaseP primers will amplify gDNA, which is why there is no loss of signal.

I can see from your paper that you managed to solve this problem. You mentioned that you planed on increasing the amount of beads? Did this work or was there any other ticks that made the difference.

Any help very much appreciated!

Cheers,

Rob

 

[Alejandro Caro-Quintero]

Dear Rob,

we try adding half and double the amount of bead solution and did not find much difference in the final Ct. One thing that really helps us was to increase Ct was the incubation of 10 min @ 60 C with Proteinase K.

We have not tested the addition of mucolytics to the extraction, but Alexander James Phillips suggested several reagents that might work https://testingmethods.<wbr />crowdicity.com/post/3193060 .

The following paper also tested the effect of several conditions and concentrations, that might be also adapted.

He, H., Li, R., Chen, Y., Pan, P., Tong, W., Dong, X., … & Yu, D. (2017). Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections. Scientific reports, 7, 45199.

Best,

Alejandro

 

 
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[Rob Page]

Thank you very much for your reply Alejandro. It’s interesting that Proteinase K 10 mins @ 60 degrees should make a difference to the RdRP CT and not the RNaseP. Do you think it is simply breaking down the mucus/proteins to make the RNA more accessible to the beads? I will give it a go with our protocols tomorrow and let you (and everyone else here) know.

Can I ask why 60 degrees? Is this based on the scientific reports paper you linked that shows lower CTs at higher temperatures (although the authors are not clear which steps of their protocol are at higher temperatures). It seems to me that this temperature is optimal for RNase activity (although the GITC and Proteinase K will protect against this).

 

[Sheila Ons]

Hi,

We are doing viral extraction by replacing GITC buffer by Trizol. Does we need to add RNA binding buffer to the samples? or can we follow the same protocol? What is RNA binding buffer used for?

Thanks a lot for your helpful answer.

 

Sheila

[Neil Ashley]

Hi,

in Oxford UK we’ve set up the Covid BOMB technique on our Beckman FxP robot and we are following the Columbian protocol with proteinase K + heating the swab samples @ 60C in a 96 deepwell plate for 30 minutes. Not including the incubation times it can do 96 samples in about 40 minutes.

Problem is we’ve seen major clumping of the beads with some of the swab material, sometimes so bad the beads don’t pellet properly and are lost. We suspect this is due to DNA precipitating onto the beads so we are going to try adding DNAse I. Anyone seen this problem too?

 

[Author]

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