@ajp1
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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.
== Technology adoption – who’s using what variant, where, for what ==
Hi all,
I went through the different forum posts to see who has said what and tried to guess where you’re all at with the Bomb.Bio protocol for RNA purification. Specifically I’m looking for technicians, scientists, or managers at public pathology labs, academic researchers, or private companies who are:
- Evaluating (the Bomb.bio RNA purification technique for clinical sample testing with or without automation)
- Deployment in progress (for clinical sample testing)
- Clinical sample testing
Please correct / add your details here: https://docs.google.com/spreadsheets/d/13_cSMSHtzMu0l1TCJF3KzCdXo88ZPCc1OqSKuAnQAQQ/edit#gid=0
It’s purpose is to:
- allow other pathology lab staff to get a sense for the adoption of the technique
- to make them feel more confident they’re going to be able to do something worthwhile
- and that there’s lots of other people / labs out there who can support them if they have problems implementing it. (I know the Bomb.bio team are very responsive on the forum anyway but they don’t necessarily know that).
Thank you!
AJPHi Rachel,
If you want there’s a Bomb.Bio / OpenTrons RNA purification protocol from OpenCell here: https://github.com/UK-CoVid19/OpentronDev/blob/master/protocols/RNA%20Extraction%20MD%20V9.py
With accompanying doc here: https://docs.google.com/document/d/11Kfc2KW56N5ggyGUDIIb-YVgKA0DsH8MjnlfujjUXmc/editGive me a shout if you need anything.
@Emily/Bradley it would be great to hear how / if you’ve progressed. Any stumbling blocks you’ve had. Where you’re based. I’m collating a list of labs experimenting with / using Bomb.Bio extraction protocol here: https://docs.google.com/spreadsheets/d/13_cSMSHtzMu0l1TCJF3KzCdXo88ZPCc1OqSKuAnQAQQ/edit?usp=sharing (I’ve added you)
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,
AJPp.s. when you are using Bomb.Bio for clinical sample processing please send a message to let everyone know 🙂
Sorry I realised I forgot to give this feedback:
While GITC + detergent buffers are expected to inactivate coronaviruses (e.g. MERS-CoV, Kumar et al., 2015 https://www.ncbi.nlm.nih.gov/pubmed/26190637 ), this has not been definitively tested for SARS-CoV-2.
Note that Kumar et al. paper actually uses GITC (guanidinium isothiocyanate) containing AVL __with ethanol__:
AVL buffer (Qiagen) at a 4:1 ratio (400 μl AVL:100 μl supernatant) and incubated at room temperature for 10 min. Ethanol (>95%) was added to a final volume of 900 μl and vortexed.
So I believe it was misleading of Kumar et al. to say AVL inactivated the MERS virus.
Cheers,
AJP
p.s. there is more conversation here (as well as in other places) on lysis buffer inactivation of SARS-CoV-2: https://testingmethods.crowdicity.com/post/3161470
Following up on: https://bomb.bio/forums/topic/the-official-sa…ocol/#post-9304
Hi Tim & team,
I’ve contacted a few NHS labs in the UK reporting shortages of RNA extraction as well as posted on the UK DHSC crowd-sourcing ideas page ( https://testingmethods.crowdicity.com/post/3163640 ). Being able to point to a peer reviewed publication would aid adoption. I’d be happy to write up the paper for CHL / your NZ team member without being credited, I just need the key data. I think this is all we’d need:
- number of patient samples
- was Bomb.bio extraction paired with a “gold standard” RNA extraction step, if so which one
- what where the Cq values from each paired run
- what RT-qPCR reagents, protocol and probes were used
- any modifications or observations on the Bomb.bio protocol.
I’m going to reach out to CHL, is there anyone there you’d recommended contacting? Thank you!
AJP
p.s. if you know of any NHS or public health labs in other countries who have published results of validating this protocol already then that would also obviously be enough.
Replying to: post-9312 “bead aggregation in IPA; timeframe and brand?”
The OpenCell team are really busy so I don’t know the full answer but they said “it was pretty immediate”.
mixed the IPA and beads by hand just before we started the run. He suspended the mixture manually so it was well mixed. Then, by the time the run started the beads were settled at the bottom of the well, and pipetting/mixing (which is the only way the OT2 can mix) didn’t work.
Not sure about bead brand. I believe they’re silica coated.
Another question / observation. Protocol says: “NB, beads and isopropanol can be premixed to improve workflow” the team at OpenCell found the “beads congregate in the IPA and can’t easily be resuspended”. Perhaps we should remove this note or qualify it with a “NB, beads and isopropanol can be premixed to improve workflow. However test before hand as sometimes they aggregate and can not easily be resuspended.”?
Do you host the latex version of these files on something like github? I’d be happy to open an issue / submit a pull request.
Will the NZ Bomb.bio and Canterbury Health Laboratories teams publish a paper on the validation of the protocol? For example:
- Including the number of patient samples that were analysed using this method
- The extraction protocol / system this was compared against
- The Ct values per sample from the comparison protocol and from this protocol
- Which supplier of commercial carboxylate coated paramagnetic beads was used
Thank you for releasing the protocol so quickly and for this amazing work. Looking forward to seeing someone validate the full protocol from sample to result using all open-source / non-proprietary reagents.
Thank you Tim. Completely agree. It’s an absolute honour to receive your message. Please contact me if you are blocked by any specific IP as currently working with an amazing bunch of people pushing for something like: http://coronaopensource.org/licences
Done a BBC interview last week (11:45+) https://www.bbc.co.uk/sounds/play/w3csy7l6 and will hopefully be talking to more press shortly so you may get press coming your way.
Hi team,
I love your commitment to open science. I’m co-founder of HelpfulEngineering.org (13,000+ volunteers against Covid-19).
Would a modified version be applicable to RNA extraction of Corona virus from samples from a human? Presumably we’d change the protocol to remove Step 2 “Pellet the cells via centrifugation at 500g and discard the supernatant”?
Many thanks,
AJP
For others looking there was an answer given to this question here: https://bomb.bio/forums/topic/modified-stober-process/#post-8975
Eunhae, it would be good to hear whether this approach worked for you?
Kind regards,
AJP
