- This topic has 27 replies, 11 voices, and was last updated June 26, 2020 at 2:27 am by .
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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.
Good question.
I don’t think anyone from BOMB is currently planning on doing so and I do not know whether Canterbury health has the capacity to go through the rigmarole of scientific publishing while providing the COVID-19 testing service at the same time. I will raise this issue with my colleagues asap.
Cheers
Tim M
Thanks Tim.
Alexander – Tim M is correct. Our focus is on delivering a diagnostic service at present. However, I will take your comments under consideration and we’ll see what our team can do.
To answer a couple of questions though:
Our validation was compared to the Abbott RealTi<i>m</i>e SARS-CoV-2 kit and extraction on an Abbott M2000. The Ct values for positive controls differ by 1-3 cycles (with the BOMB data coming up later) – this is likely due to a decrease in the amount of input VTM/UTM though, not a decrease in extraction efficiency.
We are using GE Healthcare Sera-Mag Magnetic Speedbeads (GEHE4515210).
We have not yet moved this method to any of the liquid handling systems that are currently available to us due to the logistics of sourcing appropriate plasticware. When we do, I will make sure we make our protocols available to you.
Cheers,
Kylie
Dear Admin,
It is really excellent protocol for a quick purification. It works better most cases but few samples showed one gene positive and one was negative.
Our elution buffer volume is 40 ul I wounder if we use more volume like 80 ul for eluting the RNA will it affect the detection.
I saw many automated machine are using 300 ul of VTM samples for isolation then final elution was at 150 ul then they are going 5 ul for RT-QPCR for the reaction volume of 20 ul
please give me an suggestion of sample volume to use in PCR reaction or any dilution if required. Since we are isolating total nucleic acid it might be inhibiting the reaction.
Thanks in advance!
Regards
Muthu
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.
Ahoy,
Thank you for this valuable feedback. We do not have the file on github but I will talk to my colleagues and and try to address this issue asap. Do you happen to have any information on a time-frame within which this became an issue for you? Also, what beads (manufacturer) are you using? While I have premixed isoprop and beads without any issues I have personally never done so more than a few minutes (1-5min) before sample addition.
This might sound a bit obvious but I will still mention it: I have encountered beads that settled into a somewhat firm pellet after weeks of storage (in buffer, not isoprop.) that proved to be pretty much immune to vortex. However in this case up and down shaking by hand always solved the issue within seconds.
Best Regards
Tim M
Hi all,
We have been able to test this protocol on real samples and see it work, but with less efficiency compared to spin column extraction. We’d like to keep using this protocol for kit-less automation but looking for ways to increase extraction efficiency. Does anyone know if using silica beads vs. carboxylated beads will make a difference, I notice a lot of the mag-kits use silica beads? Would they be interchange able with this protocol?
Any insights would be helpful.
Emily
I cannot tell you if you could achieve higher efficiency with carboxyl rather than silica beads.But I am quite certain, that this protocol would work with either. What you could try is diluting your stock bead concentration a little less to determine if limited bead RNA binding capacity is responsible for the lower efficiency. Furthermore no matter what beads you use, the smaller their average size the higher their capacity per weight/volume of beads.
cheers
Tim M
Hi Emily – details for the beads used in the COVID protocol can be found at the bottom of the protocol page (GE Healthcare Sera-Mag Magnetic SpeedBeads Carboxylate-Modified Dia.: 1 μm; GEHE451521 05050250) along with details on their preparation.
We’ve not tested BOMB silica beads for SARS-CoV-2 yet, however, this will happen soon. In the meantime, you might be interested in the direct comparisons between Speed Beads and BOMB silica beads we previously did for DNA extraction – there was no difference (see Figure S4 of Oberacker et al., 2019), but both were generally better than Phenol-Chloroform.
Best wishes,
TimH
Thank you for providing this protocol. If I read it correctly, the RNA is washed off the beads with just water. Is that correct? I’m wondering why some of the kits have expensive elution solutions and if water works just as well for recovery. Thank you! Ellen
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.
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.
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
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