[admin]

IMPORTANT:
The BOMB team strongly advice not to try this protocol on actual COVID-19 samples unless you were trained in all the necessary precautions required to handle highly infectious viral material. There are also legal aspects to consider when handling a notifiable disease vector. The BOMB team consists of a diverse group of scientists, none of which are Lawyers, Epidemiologists or even Virologists. Ultimately the BOMB team can only provide advice on how to extract RNA using magnetic beads.
We do not take responsibility for any accidents, infections or legal implications resulting from the use of protocols hosted on BOMB.bio.

Canterbury Health Laboratories together with members of the BOMB team have successfully trialed a protocol for the extraction of COVID-19 RNA from nasal/throat swabs that were collected in Viral Transfer Media. The protocol is based on established BOMB protocols with some minor modifications to suit collection of viral swabs and RNA purification from them.

This COVID-19 protocol is now available on the protocol page.
Or, by clicking on this link which will take you directly to the protocol PDF.

Handling of samples containing SARS-CoV-2 is associated with significant risks!

A special thanks to the people having adjusted and tested this protocol and to everyone who is helping in this global effort to quell this pandemic, thru testing, support or simply, social distancing.

Stay Safe!

[admin]

Reserved Space

[Alexander James Phillips]

Will the NZ Bomb.bio and Canterbury Health Laboratories teams publish a paper on the validation of the protocol?  For example:

1. Including the number of patient samples that were analysed using this method
2. The extraction protocol / system this was compared against
3. The Ct values per sample from the comparison protocol and from this protocol
4. 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.

[admin]

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

 

[Alexander James Phillips]

Thank you Tim!  Points awarded for data and clarity.  Not for formatting and prettiness 😉 🙂

[Kylie Drake]

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

 

[pazhanimuthu Annamlai]

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

[Alexander James Phillips]

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.

[admin]

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

[Emily Junkins]

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

[admin]

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

[TimH]

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

 

[Ellen]

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

[Alexander James Phillips]

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.

[Alexander James Phillips]

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:

1. number of patient samples
2. was Bomb.bio extraction paired with a “gold standard” RNA extraction step, if so which one
3. what where the Cq values from each paired run
4. what RT-qPCR reagents, protocol and probes were used
5. 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.

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