DIY COVID-19 PCR test

[Tito]

Anyone else curious about this?

BioCurious did a hackathon/co-learning event 4 weeks ago (https://www.meetup.com/BioCurious/events/268159598/). Also saw the post about OpenCell (https://www.opencell.bio/biohackathons). Shenzhen Open Innovation Lab (SZOIL) just announced Hack for Wuhan (https://www.bagevent.com/event/6368833).

Tito

[Derek]

Hey Tito,

Been doing some research on this. With the long incubation time I'd like to know when it's circulating in the community before cases are seen. Have found an rt-pcr protocol and located a company that will sell me a plasmid with part of the capsid envelope to use as a positive control. Biobasic has all the primers cheaply. Still working on a collection protocol to allow me to swab banisters, etc. A bunch of folks at the victoria makerspace are interested in helping so will eventually be documenting our steps. Happy to share any details.

Derek

[William Heath]

I am interested.  I am a software engineer.  How can I contribute/help in this process?

-Tim BCSE, MSCS, MBA

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[jerry maxwell]

I work in the industry as a surplus bio/genetic equipment supplier. I was checking out the CDC recommendations and kits available for corona virus identification and it looks like older ABI equipment, such as the 9700 silver and gold thermal cyclers and the ABI 7500 Fast Analyzers are going to be in demand for this testing. It's funny because these are practically antiques now, and supply is very limited. I don't see much progress towards a vaccine until late this year... so be ready; there will be quite a demand in detection of this new pandemic.        Jerry, CSI Sonora California

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[Tito]

Please share all details, would love to hear more as I'm sure others would. You are likely the first person in the world to do this at a DIY level.

[Tito]

Links to share? Would be interested in hearing what part of it implies such out of date equipment.

[Thomas Landrain]

Hi there from Paris, France,

Great initiative! 

I’d love to help organize a community of contributors aiming to design a DIY 2019-nCoV diagnosis test. I’m a biologist myself (been in this beautiful community for 11 years now (: ) and I’m running an open source platform called Just One Giant Lab (JOGL.io) whose goal is to (1) facilitate the creation and documentation of open scientific research projects and to (2) connect impactful projects to volunteering contributors who possess relevant skills. 

I also believe we should start organizing our DIYbio community around this very goal and research various ways to provide DIY/cheap 2019-nCoV testing abilities and methodologies that are well documented and that can be reviewed by the international community too. 

Let me know if you like this idea and if yes, we can start documenting the projects on JOGL and mobilize people around them. 

Thomas 

On 29 Feb 2020 at 00:55 +0100, Tito <titoja...@gmail.com>, wrote:

Links to share? Would be interested in hearing what part of it implies such out of date equipment.

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[Maria Chavez]

Hey Derek,

So I did a mental walk through of doing some RT PCR sort of testing but there are many big question for me.

-  what value does this give?  The two reasons for testing is to identify it for treatment and containment.  Community labs dont have the ability to treat or training in dealing with people with positive infections who might come in so it could make them a place highly likely to put the community lab members at risk, for epidemiology its needed to have central tracking so where does this data go to?

- are the primers any good? I know the WHO tests seem to the better ones, the CDC initial test was a multi disease test that didnt work right so they are in the midst of switching systems, and need to centralize where the data is kept for tracking of outbreaks.  

- What are the possibilities for safety considerations?

Overall I want to find ways for the DIY bio community to serve their communities during such times but I also want to make sure we keep ourselves and communities safe.  

Thoughts on this?

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[Jonathan Cline]

This is a terrible idea.  No one in a DIY community should be actively going within 50' of someone who wants a covid swab test.  This is a highly contagious virus.  It is not something you want to do for the LoL's or the rep's.  The first medical doctor (and others, probably) who treated patients in China is now dead himself.  It is not something you want to do in a community environment, where, once you are infected from the DIY patient solicitations, you also infect others in the ad hoc unprotected community lab, since you may not show outward symptoms for a week or more.  Do some critical thinking, eh?   This is not the DIY project you are looking for.

On Friday, February 28, 2020 at 4:20:57 PM UTC-8, Thomas Landrain wrote:

Hi there from Paris, France,

Great initiative! 

I’d love to help organize a community of contributors aiming to design a DIY 2019-nCoV diagnosis test. I’m a biologist myself

... 

I also believe we should start organizing our DIYbio community around this very goal and research various ways to provide DIY/cheap 2019-nCoV testing abilities and methodologies that are well documented and that can be reviewed by the international community too. 

Let me know if you like this idea and if yes, we can start documenting the projects on JOGL and mobilize people around them. 

-- 

## Jonathan Cline

## jcl...@ieee.org

## Mobile: +1-805-617-0223

########################

 

[Thomas Landrain]

I think we all agree on not transforming DIYbio labs into local diagnosis medical labs for contagious diseases. Way too dangerous and irresponsible :/

However, I’m seeing an opportunity for this community to perform “safe” research projects to develop cheap and easily implementable methodologies to detect the presence of the virus. Such as the one proposed by Derek where he uses a small piece of its DNA sequence. 

The approach would be the same as for the open insuline project, the research can be done safely, but the implementation has to be done in a controlled environment which cannot obviously be DIY labs. The goal is to obtain scientific and technological commons that can be used and improved by the global community, medical scientists and international health organizations included. 

BioCurious, OpenCell and Derek’s pioneering efforts in trying to contribute to understanding and fighting Covid-19 need to be continued and this crisis is an important opportunity to mobilize safely our community around it. 

Thomas 

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[Derek]

To be clear I am in no way advocating testing of possible infected individuals in a community lab setting.

My primary goal here is to do environmental testing. Right now there is a lot of fear around this virus and there is a high likelihood that it is not yet present in most of our communities. I fear by the time it is actually present that response fatigue will have set in and people will have become more lax in self-protection. With the long latency period, while virus is being shed, it is likely that sampling of public banisters, buses, etc. may show evidence of community spread of the virus prior to showing active cases in any particular area. This would be an indication of the need to take stronger self-protective measures or at least get an updated "go to the hospital and get tested if you're ill" message out there.

In terms of primers and evidence of their working, during the initial calibration of the test I expect will have to spread some of the positive control on surfaces in the lab and see if they can be picked up, sensitivity, etc.

In terms of safety considerations, I think that this is helpful to public safety and the safety of individuals involved in the effort. Since we are discussing sampling of public spaces that are generally presumed safe, or at least safe pending mitigation such as frequent hand washing, it would seem that no additional safety risk is presented. And the act of sampling itself increases awareness of the possible vectors for infection. If we included primers for something more common, rhinovirus for instance, that we could expect to see in many places it would further the awareness of which surfaces tend to promote contagion.

It would be great if there were a test that people could do at home if they felt that they were infected, but as noted above that could have nothing to do with infected individuals or their samples entering a community lab. One approach that is promising is the dipstick-based isothermal amplification approach coming out of the Zhang lab. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf

In terms of links, I am collecting others and will update but here are a couple of the sources I mentioned:

https://www.biobasic.com/coronavirus-covid-19-primers/

https://www.molecularcloud.org/How-to-detect-the-2019-novel-coronavirus.html

https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html

and a couple different kit approaches:

https://www.idtdna.com/pages/landing/coronavirus-research-reagents

https://www.sciencellonline.com/PS/7038.pdf

Derek

On Saturday, 29 February 2020 00:20:49 UTC-8, Thomas Landrain wrote:

I think we all agree on not transforming DIYbio labs into local diagnosis medical labs for contagious diseases. Way too dangerous and irresponsible :/

However, I’m seeing an opportunity for this community to perform “safe” research projects to develop cheap and easily implementable methodologies to detect the presence of the virus. Such as the one proposed by Derek where he uses a small piece of its DNA sequence. 

The approach would be the same as for the open insuline project, the research can be done safely, but the implementation has to be done in a controlled environment which cannot obviously be DIY labs. The goal is to obtain scientific and technological commons that can be used and improved by the global community, medical scientists and international health organizations included. 

BioCurious, OpenCell and Derek’s pioneering efforts in trying to contribute to understanding and fighting Covid-19 need to be continued and this crisis is an important opportunity to mobilize safely our community around it. 

Thomas 

On 29 Feb 2020 at 08:23 +0100, Jonathan Cline <jnc...@gmail.com>, wrote:

This is a terrible idea.  No one in a DIY community should be actively going within 50' of someone who wants a covid swab test.  This is a highly contagious virus.  It is not something you want to do for the LoL's or the rep's.  The first medical doctor (and others, probably) who treated patients in China is now dead himself.  It is not something you want to do in a community environment, where, once you are infected from the DIY patient solicitations, you also infect others in the ad hoc unprotected community lab, since you may not show outward symptoms for a week or more.  Do some critical thinking, eh?   This is not the DIY project you are looking for.

On Friday, February 28, 2020 at 4:20:57 PM UTC-8, Thomas Landrain wrote:

Hi there from Paris, France,

Great initiative! 

I’d love to help organize a community of contributors aiming to design a DIY 2019-nCoV diagnosis test. I’m a biologist myself

... 

I also believe we should start organizing our DIYbio community around this very goal and research various ways to provide DIY/cheap 2019-nCoV testing abilities and methodologies that are well documented and that can be reviewed by the international community too. 

Let me know if you like this idea and if yes, we can start documenting the projects on JOGL and mobilize people around them. 

-- 

## Jonathan Cline

## jcl...@ieee.org

## Mobile: +1-805-617-0223

########################

 

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[Derek]

Forgot a reference. Not covid, but a study of surfaces in an airport that shows an analagous approach.

https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-018-3150-5

[Tito]

Hi Maria,

Great questions here. For those catching up, Maria hosted a Wuhan Virus Co-Learning Hackathon on Feb 1st, plus she's the president of BioCurious so she has lots of firsthand experience here. I just posted another thread to share out insights from those 2 events here.

What is the value of community labs in a COVID-19 world?

Maria and Jonathan Cline above make great points about the challenges of working with samples.

Perhaps the biggest outcome is that everyone decides *not* to work on this at a PCR level and each lab does a blog post on it. Or spend energies elsewhere like working on the genome virtually, software to map spread of the virus like http://outbreak.cc/, building/testing sterilization protocols, hygiene inventions like copper infused gloves, testing out DIY face mask designs. Lots of possibilities here.

Tito

On Friday, February 28, 2020 at 4:43:00 PM UTC-8, Maria Chavez wrote:

Hey Derek,

So I did a mental walk through of doing some RT PCR sort of testing but there are many big question for me.

-  what value does this give?  The two reasons for testing is to identify it for treatment and containment.  Community labs dont have the ability to treat or training in dealing with people with positive infections who might come in so it could make them a place highly likely to put the community lab members at risk, for epidemiology its needed to have central tracking so where does this data go to?

- are the primers any good? I know the WHO tests seem to the better ones, the CDC initial test was a multi disease test that didnt work right so they are in the midst of switching systems, and need to centralize where the data is kept for tracking of outbreaks.  

- What are the possibilities for safety considerations?

Overall I want to find ways for the DIY bio community to serve their communities during such times but I also want to make sure we keep ourselves and communities safe.  

Thoughts on this?

On Fri, Feb 28, 2020 at 9:19 AM Derek <der...@gmail.com> wrote:

Hey Tito,

Been doing some research on this. With the long incubation time I'd like to know when it's circulating in the community before cases are seen. Have found an rt-pcr protocol and located a company that will sell me a plasmid with part of the capsid envelope to use as a positive control. Biobasic has all the primers cheaply. Still working on a collection protocol to allow me to swab banisters, etc. A bunch of folks at the victoria makerspace are interested in helping so will eventually be documenting our steps. Happy to share any details.

Derek

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[Jonathan Cline]

Smartwatch app to periodically measure body temperature and sync to
the cloud with location data for crowdsourcing potential outbreaks
would be more relevant I would assume. WHO should already have
developed such an app but they're obviously behind the times. The
drawback being that smartwatch temperature sensors have improper reads
sometimes.

Developing a test kit for swabbing potentially contaminated surfaces
is not much safer than swabbing potential patients themselves.

Benefits of a face mask are nebulous. They certainly don't protect
against a virus and maybe only marginally protect others when an
infected person wears one.

More useful could be a (pressure) directed ozone generator to
sterilize public surfaces, portable size, with a rechargeable battery
pack.

On 2/29/20, Tito <titoja...@gmail.com> wrote:
>

> Perhaps the biggest outcome is that everyone decides *not* to work on this
> at a PCR level and each lab does a blog post on it. Or spend energies
> elsewhere like working on the genome virtually, software to map spread of
> the virus

[Derek]

Absolutely disagree on the relative risk of surface swabbing. Presumably if individuals are passing through the areas that they are swabbing they are picking up these pathogens on their hands anyway. The additional risk of swabbing is negligible. Back in the lab, the samples have to be presumed to to be infectious, but no amplification of the entire virus is occurring. The PCR step is amplifying only a portion of the envelope. Slight risk of aerosolizing the particles, but nowhere near the risk of an individual who is infected aerosolizing particles just by breathing, let alone coughing.

[Tyler Quarton]

Hi Maria,

I'm currently developing a platform that facilitates data sharing and collection for citizen scientists. I told my development team about this diy covid initiative and we are now working rapidly to get a minimally functioning prototype in hopes to create that centralized database for tracking. I'll keep the group in the loop as we move forward. 

On Friday, February 28, 2020 at 7:43:00 PM UTC-5, Maria Chavez wrote:

Hey Derek,

So I did a mental walk through of doing some RT PCR sort of testing but there are many big question for me.

-  what value does this give?  The two reasons for testing is to identify it for treatment and containment.  Community labs dont have the ability to treat or training in dealing with people with positive infections who might come in so it could make them a place highly likely to put the community lab members at risk, for epidemiology its needed to have central tracking so where does this data go to?

- are the primers any good? I know the WHO tests seem to the better ones, the CDC initial test was a multi disease test that didnt work right so they are in the midst of switching systems, and need to centralize where the data is kept for tracking of outbreaks.  

- What are the possibilities for safety considerations?

Overall I want to find ways for the DIY bio community to serve their communities during such times but I also want to make sure we keep ourselves and communities safe.  

Thoughts on this?

On Fri, Feb 28, 2020 at 9:19 AM Derek <der...@gmail.com> wrote:

Hey Tito,

Been doing some research on this. With the long incubation time I'd like to know when it's circulating in the community before cases are seen. Have found an rt-pcr protocol and located a company that will sell me a plasmid with part of the capsid envelope to use as a positive control. Biobasic has all the primers cheaply. Still working on a collection protocol to allow me to swab banisters, etc. A bunch of folks at the victoria makerspace are interested in helping so will eventually be documenting our steps. Happy to share any details.

Derek

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[Dakota Hamill]

What's the platform? Can we beta test it or is it launched? 

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[Michael Crone]

In my opinion the Zhang lab post is not great. It ignores everything that would actually make it clinically relevant (e.g. sample processing). It’s completely unrealistic to give people the idea that you can just have a viral load high enough to do a SHERLOCK assay without any sample processing. And the sample processing is the most difficult part. Their intention seems to be just to get a little bit of PR.

There are many issues when dealing with the virus that need to be considered. The current testing is only around 40% accurate because the viral load from throat swabs is unreliable. Even then, you need to have a sample processing step before you do the RT-PCR. The next problem is scaling. Most laboratories are not equipped to perform thousands of tests in a day. In China, regular biology labs at universities are doing the testing and they couldn’t do more than 2000 a day with 50 staff. This is because sample processing is done in a P3 biosafety setting. RT-qPCR is just not readily scaleable and there will also be massive problems when African countries have to try and test large portions of the population. The current mainstays (Roche and Abbott) can only perform around 1000 tests in an automated fashion in a shift and they are dependent on their own reagents.

When trying to do community led research it needs to be planned out. Adding a bit of plasmid to a lab bench and then seeing if you can amplify it ignores many of the aspects of the virus that make it clinically relevant. What’s a relevant concentration? How does DNA plasmid compare to an RNA virus? How stable is DNA compared to RNA? How do you know how many copies you’re adding to the lab bench (260/280 is horribly unreliable when determining copy number)? What are really the questions that you would like to answer and how would they make a difference?

[S James Parsons Jr]

Hi Tyler,

I’m interested in talking with you on your data sharing platform, do you have a website with more information?  I too am working on software for citizen science to share and collaborate 

James

Sent from my iPhone

On Feb 29, 2020, at 9:05 PM, Tyler Quarton <tqua...@gmail.com> wrote:



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[Derek]

The real question in my mind was to assess whether there was community spread in advance of seeing clinical cases. Things may be moving too fast for that to be relevant, though, as the sequencing data shows some pretty compelling evidence that it is already in community spread now. At least in Western Washington which is so close to me in Victoria, BC that it is undoubtedly here as well. https://twitter.com/trvrb/status/1233970271318503426

This time lag between when spread begins and when cases are seen clinically is the biggest barrier to control in my view. The fact that the Washington evidence suggests it has been in community circulation for 6 weeks there already implies that containment may unfortunately be a lost cause.

[Reginald Smith]

If you get these working let us know. It seems that it is a huge cocktail with the first two primer pairs (1-4) in the BioBasic kit being the ones originally developed and recommended by the Chinese government in late January, the last four (9-12) are two pairs from the WHO recommendations and (5-11) it is unclear where they are from (no Google matches). Maybe they developed independently at BioBasic using conserved genome regions. Interesting the CDC recommended primers aren't in there. I have seen so many different primer sets, some with huge overlap in the coronavirus family, that I am not sure if a gold standard is agreed upon yet. I'm sure they all work to some extent though.

Reggie

[Michael Crone]

It's possible that I could provide an RNA standard that I have developed for the coronavirus (based on using MS2 phage as a cage for packaged, something called "Armoured RNA"). At the moment it is just the N gene, but I am planning to make for the Env gene as well (depending on what route the diagnostic herd goes). Just not sure how sharing works outside of the research community (or if I would be allowed to share it).

It would probably serve as a much better standard than just using plasmid and it is more stable than a plasmid too I think.

Michael

[AJP]

I'd like to be able to test myself.  I'm cancelling events because I have had a tiny dry throat for 3 days that's almost certainly a "normal" seasonal bug.  The local advice for us is to self isolate if unsure / you're probably fine at the moment.  Neither of these are satisfying for me.  These approaches also don't help as I will need to buy food sooner or later and I'd like to go to the shops when I'm not contagious but I only have 3 weeks food supply so it's not possible to just sit at home for now.

It would be great to be able to provide an open source solution for 1000s of people out there who want to test themselves or offer to test people they can't avoid being around like their family, friends and co-workers.  If we're going to be sat next to them any way then it would be great to know that, as of this morning, your viral load is undetectable through this particular protocol / tool combo.

Thoughts, criticism, agreement welcomed.

With kind regards,

AJP

[AJP]

Hi Michael,

I might has misinterpreted what Derek was saying here and what what you've said.  However I think Derek's

plasmid with part of the capsid envelope to use as a positive control

was referring to the need to have a positive control when trying to use PCR on a sample.  Not to, as you put it, 

Adding a bit of plasmid to a lab bench

I think Derek was suggesting taking samples in his environment, that he'd be exposed to anyway, and then testing them for the presence of the virus.  Using the plasmid as a positive control.  Apologies if I've misinterpreted what you were saying.  Broadly speaking I agree with what you've said.

With kind regards,

AJP

[AJP]

Hi Derek,

What company is that?  And how much / what's their lead time?  I'm asking as some of us from the London BioHackSpace want to be able to test ourselves and those near to us... to minimise us infecting others if / when we're infected.

Thank you,

AJP

p.s. if you're interested you can join the slack group and join the #lets-beat-corona channel , though we'll be sure to post here with any relevant info / decisions / purchases.

[Jonathan Cline]

On 3/4/20, AJP <bio...@gmail.com> wrote:
> I'd like to be able to test myself.

Great. Except for the danger of the very likely false negatives.

[Thomas Landrain]

Hi there, 

We launched an OpenCovid19 Initiative on JOGL to collectively design a safe open source DIY Covid19 diagnostic test. 

You are welcome to join, 25 people already joined in 72h! 

More info here: https://app.jogl.io/project/118

We are having our first community conference call today at 9pm Paris time (https://zoom.us/j/7804345815)

Cheers

Thomas

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Co-Founder & President, JOGL.io - Just One Giant Lab

Member, Conseil National du Numerique

Ambassador, iGEM Foundation

Co-Founder & former CEO, PILI

Co-Founder & former CEO, La Paillasse

Twitter: @Tholand_ @JustOneGiantLab @_PILIbio

Mobile: +33 678 37 31 36

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[AJP]

tl, dr; if you get a DIY positive result I think that's useful, if it's negative then assuming everything else is equal you're in no different position to everyone else who hasn't been tested.

danger of the very likely false negatives

Yes, I absolutely agree Jonathan; false negatives are a problem.  With the use of a positive control plasmid Derek mentioned you can reduce some of the sources of false negatives such as completely incorrect or damaged primers, wrong temperatures for annealing or elongation, correct temperatures but faulty hardware, contamination of reagents with DNA nucleases (doesn't help with RNA specific nucleases), non functional polymerase, not adding NTPs, otherwise faulty master mix, not loading the gel correctly (assuming not using rtPCR), not reading it correctly, not running it correctly, not using the correct buffers, contamination with DNA nucleases, etc.  You can't correct for false negative error from inadequate DNA extraction protocol, not following extraction protocol, RNA specific nuclease contamination, non matching primers for different strain, etc.

Successful DNA extraction from samples and PCR in DIY settings though have and are performed.  I would not trust a negative DIY result if I had symptoms or reason to believe I was infected, but I definitely would not assume I was free from the virus if I had a positive result but no symptoms or reason to believe I was infected.

This is based on the assumption that the rate of false positives is small and that false negatives are the main source of error.  If you do get a negative then I think you're no worse off and are back to where you were before; do you disagree?  I've tried to explore what we might do differently given different DIY results (please add your comments or edit as you wish), assuming again that you're even moderately sure false positives are minimised because otherwise I think there's very little point in doing it in the first place.

Cheers,

AJP

[AJP]

I think this is good to point out.  I was not imagining testing anyone else apart from myself.  Tom summarises it well in this JOGL intro video for project 118.  Though I think environmental sampling could increase your risk: I don't normally touch anything with my hands on public transport... it's elbows all the way.  But if you usually hold onto hand rails then you're probably no worse off wiping it with some polyester wipe before you do.

AJP

[Jonathan Cline]

Would someone post the datasheet(s) of the existing biopharma tests?
There have been various news reports of commercial test failures.

Quote from news a few weeks ago: "It remains unclear whether the CDC’s
move on Wednesday will resolve all of the problems around the test.
Some local labs have raised concerns about whether the CDC’s test is
fully reliable for detecting COVID-19. In New York, scientists at
both the city’s and state’s laboratories have seen false positives
even when following the CDC’s latest directions, according to a person
familiar with their discussions."

An erroneous test result can probably be shown to be worse than having
no result at all. I am sure diybio will forge ahead independent of
that.

[Reginald Smith]

I agree, there are a LOT of primers floating out there. The only attempt at comparison for validation I found was the below from S. Korea. FYI, everyone should know BiorXiv only accepts papers that are not peer reviewed (yet) so this isn't an endorsement..

Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is newly emerging human infectious diseases, which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within two months of the outbreak, more than 80,000 cases of COVID-19 have been confirmed worldwide. Since the human to human transmission occurred easily and the human infection is rapidly increasing, the sensitive and early diagnosis is essential to prevent the global outbreak. Recently, World Health Organization (WHO) announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA. In this study, we compared the ability to detect SARS-CoV-2 RNA among the seven primer-probe sets for N gene and the three primer-probe sets for Orf1 gene. The result of the comparative analysis represented that the ‘2019-nCoV_N2, N3’ of USA and the ‘ORF1ab’ of China are the most sensitive primer-probe sets for N and Orf1 genes, respectively. Therefore, the appropriate combination from ORF1ab (China), 2019-nCoV_N2, N3 (USA), and NIID_2019-nCOV_N (Japan) sets should be selected for the sensitive and reliable laboratory confirmation of SARS-CoV-2.

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[Tito]

U Washington has its own test:
https://mobile.twitter.com/uwmnewsroom/status/1235630636913709057?s=21

[Michael Crone]

Ct value is the number of cycles before you get above a certain threshold fluorescence. Lower means more sensitive in the context of the paper.

There is a massive issue with everyone developing their own tests because it just causes massive fragmentation. Ideally researchers should be working towards implementation solutions. Developing tests is easy, actually implementing them in a clinical context is the difficult part.

Michael

On Fri, 6 Mar 2020 at 16:42, AJP <bio...@gmail.com> wrote:

Derek had replied here linking to: https://www.molecularcloud.org/How-to-detect-the-2019-novel-coronavirus.html

On a separate note does anyone know what "Ct value" means in the context of this paper please?

Results and Discussion
Validation of qRT-PCR assay
The Ct value was not produced from negative control, indicating the reaction was done aseptically.

Thank you,

AJP

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[Jonathan Cline]

https://www.epa.gov/sites/production/files/2020-03/documents/sars-cov-2-list_03-03-2020.pdf

EPA’s Registered Antimicrobial Products for Use Against Novel
Coronavirus SARS-CoV-2, the Cause of COVID-19

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460
OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION
03/03/2020

[AJP]

Thank you for the response Michael.  I had deleted my post because I hadn't searched for "Ct value" properly (was the top hit for "RT PCR Ct value")

I don't have any firsthand knowledge of the challenges associated with "working towards [clinical] implementation solutions" so I can't comment on this.

Hopefully myself or those close won't be infected (if there are statistically we look safe) but if so then trying to replicate that same paper might prove interesting / be useful.  There will unfortunately be differences though as I don't have access to a RT-PCR.  Instead I'll look for the appropriate sized bands on a gel.

Kind regards,

AJP

On Friday, March 6, 2020 at 4:48:23 PM UTC, Michael Crone wrote:

Ct value is the number of cycles before you get above a certain threshold fluorescence. Lower means more sensitive in the context of the paper.

There is a massive issue with everyone developing their own tests because it just causes massive fragmentation. Ideally researchers should be working towards implementation solutions. Developing tests is easy, actually implementing them in a clinical context is the difficult part.

Michael

On Fri, 6 Mar 2020 at 16:42, AJP <bio...@gmail.com> wrote:

Derek had replied here linking to: https://www.molecularcloud.org/How-to-detect-the-2019-novel-coronavirus.html

On a separate note does anyone know what "Ct value" means in the context of this paper please?

Results and Discussion
Validation of qRT-PCR assay
The Ct value was not produced from negative control, indicating the reaction was done aseptically.

Thank you,

AJP

 

On Wednesday, March 4, 2020 at 5:51:25 PM UTC, AJP wrote:

Hi Derek,

What company is that?  And how much / what's their lead time?  I'm asking as some of us from the London BioHackSpace want to be able to test ourselves and those near to us... to minimise us infecting others if / when we're infected.

Thank you,

AJP

p.s. if you're interested you can join the slack group and join the #lets-beat-corona channel , though we'll be sure to post here with any relevant info / decisions / purchases.

On Friday, February 28, 2020 at 5:19:20 PM UTC, Derek wrote:

Hey Tito,

Been doing some research on this. With the long incubation time I'd like to know when it's circulating in the community before cases are seen. Have found an rt-pcr protocol and located a company that will sell me a plasmid with part of the capsid envelope to use as a positive control. Biobasic has all the primers cheaply. Still working on a collection protocol to allow me to swab banisters, etc. A bunch of folks at the victoria makerspace are interested in helping so will eventually be documenting our steps. Happy to share any details.

Derek

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[Kari Paul]

Hello, I am a journalist with the Guardian hoping to write about folks seeking DIY/hacking solutions to COVID-19. Would anyone here be willing to discuss this week? Let me know.

You can reply here or contact me directly at kari...@theguardian.com

Thanks!

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[Jonathan Cline]

Journalists are not competent nor objective. No one should talk to them.

Here are obvious prevention aspects of the story which journalists
have missed due to lack of science education and critical thinking
skills (plus alternatively, biased fear mongering and sensationalism),
presented as fact based. Any generic undergraduate who wrote articles
similar to the typical journalist for even a lower division science
class would earn a failing grade.

Evaluation of green tea extract as a safe personal hygiene against
viral infections
doi: 10.1186/s13036-017-0092-1

Effect of Tea Catechins on Influenza Infection and the Common Cold
with a Focus on Epidemiological/Clinical Studies
doi: 10.3390/molecules23071795

Disinfecting the iPad: evaluating effective methods.
doi: 10.1016/j.jhin.2014.01.012

Degree of Bacterial Contamination of Mobile Phone and Computer
Keyboard Surfaces and Efficacy of Disinfection with Chlorhexidine
Digluconate and Triclosan to Its Reduction.
doi: 10.3390/ijerph15102238

Persistence of coronaviruses on inanimate surfaces and their
inactivation with biocidal agents.
doi: 10.1016/j.jhin.2020.01.022


The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest
and biggest global health threats: what lessons have we learned?
doi: 10.1093/ije/dyaa033

CONCLUSIONS: We conclude that we did not learn from the two prior
epidemics of coronavirus and were ill-prepared to deal with the
challenges the COVID-19 epidemic has posed. Future research should
attempt to address the uses and implications of internet of things
(IoT) technologies for mapping the spread of infection.

On 3/9/20, 'Kari Paul' via DIYbio <diy...@googlegroups.com> wrote:
> Hello, I am a journalist with the

[Sean Rowshandel]

People are telling the media (https://www.latimes.com/science/story/2020-03-12/why-does-it-take-so-long-to-make-a-coronavirus-vaccine) the only type of globally-scalable vaccine would enter the nucleus by viral vector. There has been a nonviral vector around for decades without the side effects viral vectors have today. Using ORMOSIL (organically modified silica) instead of a viral vector would reduce the time it takes to find out a vaccine is safe (the yearlong process of which is itself an enormous tradeoff as opposed to creating whatever we can THIS WEEK to fight Covid-19, while simultaneously starting on longer-term projects to create better solutions).

https://www.researchgate.net/publication/6306992_Nonviral_gene_delivery_Thinking_of_silica

https://www.sciencedirect.com/science/article/pii/S0165027007002786

2nd link describes the process of using ormosil (stir, wait 20 minutes at room temp, then inject)

On Saturday, February 29, 2020 at 5:59:29 AM UTC-5, Derek wrote:

To be clear I am in no way advocating testing of possible infected individuals in a community lab setting.

My primary goal here is to do environmental testing. Right now there is a lot of fear around this virus and there is a high likelihood that it is not yet present in most of our communities. I fear by the time it is actually present that response fatigue will have set in and people will have become more lax in self-protection. With the long latency period, while virus is being shed, it is likely that sampling of public banisters, buses, etc. may show evidence of community spread of the virus prior to showing active cases in any particular area. This would be an indication of the need to take stronger self-protective measures or at least get an updated "go to the hospital and get tested if you're ill" message out there.

In terms of primers and evidence of their working, during the initial calibration of the test I expect will have to spread some of the positive control on surfaces in the lab and see if they can be picked up, sensitivity, etc.

In terms of safety considerations, I think that this is helpful to public safety and the safety of individuals involved in the effort. Since we are discussing sampling of public spaces that are generally presumed safe, or at least safe pending mitigation such as frequent hand washing, it would seem that no additional safety risk is presented. And the act of sampling itself increases awareness of the possible vectors for infection. If we included primers for something more common, rhinovirus for instance, that we could expect to see in many places it would further the awareness of which surfaces tend to promote contagion.

It would be great if there were a test that people could do at home if they felt that they were infected, but as noted above that could have nothing to do with infected individuals or their samples entering a community lab. One approach that is promising is the dipstick-based isothermal amplification approach coming out of the Zhang lab. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf

In terms of links, I am collecting others and will update but here are a couple of the sources I mentioned:

https://www.biobasic.com/coronavirus-covid-19-primers/

https://www.molecularcloud.org/How-to-detect-the-2019-novel-coronavirus.html

https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html

and a couple different kit approaches:

https://www.idtdna.com/pages/landing/coronavirus-research-reagents

https://www.sciencellonline.com/PS/7038.pdf

Derek

On Saturday, 29 February 2020 00:20:49 UTC-8, Thomas Landrain wrote:

I think we all agree on not transforming DIYbio labs into local diagnosis medical labs for contagious diseases. Way too dangerous and irresponsible :/

However, I’m seeing an opportunity for this community to perform “safe” research projects to develop cheap and easily implementable methodologies to detect the presence of the virus. Such as the one proposed by Derek where he uses a small piece of its DNA sequence. 

The approach would be the same as for the open insuline project, the research can be done safely, but the implementation has to be done in a controlled environment which cannot obviously be DIY labs. The goal is to obtain scientific and technological commons that can be used and improved by the global community, medical scientists and international health organizations included. 

BioCurious, OpenCell and Derek’s pioneering efforts in trying to contribute to understanding and fighting Covid-19 need to be continued and this crisis is an important opportunity to mobilize safely our community around it. 

Thomas 

On 29 Feb 2020 at 08:23 +0100, Jonathan Cline <jnc...@gmail.com>, wrote:

This is a terrible idea.  No one in a DIY community should be actively going within 50' of someone who wants a covid swab test.  This is a highly contagious virus.  It is not something you want to do for the LoL's or the rep's.  The first medical doctor (and others, probably) who treated patients in China is now dead himself.  It is not something you want to do in a community environment, where, once you are infected from the DIY patient solicitations, you also infect others in the ad hoc unprotected community lab, since you may not show outward symptoms for a week or more.  Do some critical thinking, eh?   This is not the DIY project you are looking for.

On Friday, February 28, 2020 at 4:20:57 PM UTC-8, Thomas Landrain wrote:

Hi there from Paris, France,

Great initiative! 

I’d love to help organize a community of contributors aiming to design a DIY 2019-nCoV diagnosis test. I’m a biologist myself

... 

I also believe we should start organizing our DIYbio community around this very goal and research various ways to provide DIY/cheap 2019-nCoV testing abilities and methodologies that are well documented and that can be reviewed by the international community too. 

Let me know if you like this idea and if yes, we can start documenting the projects on JOGL and mobilize people around them. 

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## jcl...@ieee.org

## Mobile: +1-805-617-0223

########################

 

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[Reginald Smith]

FYI, for those interested in this topic, a team at Yale on Wednesday released an evaluation of the SARS-CoV-2 primer sets out of the US, China, Hong Kong University, and Germany. The results are interesting though this is a MedRxiv paper that hasn't gone through peer review yet. I am sure it will review quickly due to the urgency of this issue.

For those interested in the "false negative" reports in the news recently I have not read into all the details on that but the paper reports a lower detection limit of 100 SARS-CoV-2 genome equivalents per uL. Not way off from other PCR amplification requirements but if swabbing/RNA extraction is not optimal I can see where the issues start.

In short, all the primers could detect the viral RNA but some of the primers were prone to being unable to properly distinguish between low levels of viral RNA (less than 100 genome equivalents per uL) and controls with no viral RNA. So these could theoretically give a false positive since results from no virus and low virus concentration are both below the detection cutoff for "positive" results (qPCR CT<40). This was an issue for the China primers and two of the CDC primers (N2 and N3, the latter which has already been taken out of the kit by the CDC a month ago due to the problems it was causing). The Hong Kong University primers performed the best and did not give false positive issues.

Reggie

[Reginald Smith]

Sorry I linked the wrong paper below. That was for the LAMP color detection protocol. Sensitivity paper is here

Reggie.

[Michael Crone]

I would not read too much into the paper. There was a much better Korean paper released a while ago comparing different primer sets (https://www.biorxiv.org/content/10.1101/2020.02.25.964775v1.full.pdf). The "background amplification" that would cause this in N2 and N3 seems like it could just be contamination and they don't have a proper explanation as to why this would happen (it could potentially occur with primer-probe interaction too, but Occam's razor). With a primer-probe set you really shouldn't see any fluorescent signal unless you amplify your specific target, for SYBR green yes, but probes no. Additionally, spiking raw RNA onto nasopharyngeal swabs and then drawing conclusions based on that is just not very good science. We've got down to 2.5 ul copies per reaction in our qPCR. Please don't believe anything in that paper... peer review is there for a reason.

Michael

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[boj Ko]

This is scum ... normal companies have clear rules for filling out special forms for participants who want to participate in this type.

[Reginald Smith]

Thanks, I am not super experienced in the subtleties of qPCR evaluation since I just have an old school PCR and gel. I thought it would be relevant to those here talking about testing surfaces, etc. for viral RNA which surely has concentrations much lower than the threshold the paper describes. I had read the Korean paper you refer to a couple of weeks back. I remember they were most favorable towards N2 and the Japanese primer for the N gene (both of which basically overlap the same area on the N gene within a couple dozen bp I think). Also the Orf1ab primer from the Chinese CDC.

Could I ask which primer sets you are using on your qPCR? One thing I find interesting is the CDC seems to be the only test just using one gene, the N gene. Not sure if there is a reason for this such as number of relatively conserved regions, etc.

Reggie

On Saturday, April 4, 2020 at 6:30:14 AM UTC-4, Michael Crone wrote:

I would not read too much into the paper. There was a much better Korean paper released a while ago comparing different primer sets (https://www.biorxiv.org/content/10.1101/2020.02.25.964775v1.full.pdf). The "background amplification" that would cause this in N2 and N3 seems like it could just be contamination and they don't have a proper explanation as to why this would happen (it could potentially occur with primer-probe interaction too, but Occam's razor). With a primer-probe set you really shouldn't see any fluorescent signal unless you amplify your specific target, for SYBR green yes, but probes no. Additionally, spiking raw RNA onto nasopharyngeal swabs and then drawing conclusions based on that is just not very good science. We've got down to 2.5 ul copies per reaction in our qPCR. Please don't believe anything in that paper... peer review is there for a reason.

Michael

On Fri, 3 Apr 2020 at 13:00, Reginald Smith <rsm...@supremevinegar.com> wrote:

FYI, for those interested in this topic, a team at Yale on Wednesday released an evaluation of the SARS-CoV-2 primer sets out of the US, China, Hong Kong University, and Germany. The results are interesting though this is a MedRxiv paper that hasn't gone through peer review yet. I am sure it will review quickly due to the urgency of this issue.

For those interested in the "false negative" reports in the news recently I have not read into all the details on that but the paper reports a lower detection limit of 100 SARS-CoV-2 genome equivalents per uL. Not way off from other PCR amplification requirements but if swabbing/RNA extraction is not optimal I can see where the issues start.

In short, all the primers could detect the viral RNA but some of the primers were prone to being unable to properly distinguish between low levels of viral RNA (less than 100 genome equivalents per uL) and controls with no viral RNA. So these could theoretically give a false positive since results from no virus and low virus concentration are both below the detection cutoff for "positive" results (qPCR CT<40). This was an issue for the China primers and two of the CDC primers (N2 and N3, the latter which has already been taken out of the kit by the CDC a month ago due to the problems it was causing). The Hong Kong University primers performed the best and did not give false positive issues.

Reggie

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[Rick Byers]

8 years after asking this group for advice, I've finally done my first viral diagnostics qPCR run at home. I used the Biomeme Franklin platform to test my family for Sars-CoV-2 and I'm convinced it's the first taste of what many people will be doing before long. Here's some details written for a general audience. Please forgive my over simplifications, I know many of you here are experts in all of this.

I also participated in a COVID Q&A with Max Perleman, co-founder of Biomeme. First I was pretty shocked there wasn't anyone else chomping at the bit to ask him about how technology like his can help massively scale access to COVID testing. But more importantly I was excited by the new device he gave a sneak peak of (at time 1:03). It uses a 9-well cartridge that does integrated sample prep. So you put in a raw sample and hit run and you get up to 27 assays in a single automated run. I'll definitely be buying this with a respiratory virus panel when it becomes available. Does it still count as "DIY" if there's essentially nothing to do but swab and press run?  :-)

My hope is that if there's any upside to the pandemic, it will be that it creates a consumer awareness and market for simple viral diagnostics. And if many people have a qPCR machine in their home and test kits are cheap, think of all the other science that could be done just as easily, as well as being so much better prepared for the next pandemic!

I would love to find out if the Biomeme kits can be effectively used with sample pooling techniques to effectively reduce the cost per sample by 10x or more (when infection prevalence is below 1%). In addition, recent research suggests that saliva may be even more sensitive and reliable as a sample source than nasopharyngeal swabs. I wonder if essential businesses could be doing regular practical mass screening of their employees with point-of-care devices like this by using saliva samples pooled 10x or more per test. 

But I know all of this is best explored by professional researchers and public health experts. But it seems to me like there may still be a role for biohackers and small companies to contribute and safely explore a broader array of ideas which which aren't getting the attention they deserve. If saliva really is more sensitive than NP swabs, why are we learning this >3 months into the pandemic? I like the suggestion in this thread about environmental sampling, has anyone actually tried it yet? At the least I could probably be sampling my mail and groceries to see if I can find any evidence of viral RNA at all (being careful not to imply that that is infectious of course).

We're all taking risks just trying to live day-to-day, might as well get some research out of it too! Thoughts?

Thanks,

   Rick

[Jonathan Cline]

As a better project, the local diybio communities could reach out to
local geographies for rounding up post-covid positive blood donors;
for example to respond to efforts like this one:

https://www.uscovidplasma.org
COVID-19 expanded access program
Plasma donors needed for treatment protocol
Researchers are testing the use of donated blood as a treatment for
people with severe coronavirus disease 2019 (COVID-19). People who've
recovered from COVID-19 have antibodies to the disease in their blood.
Doctors call this convalescent plasma. Researchers hope that
convalescent plasma can be given to people with severe COVID-19 to
boost their ability to fight the virus.