Do-It-Together SARS-CoV-2 Detective

Neither diagnosis of cases of Covid-19, caused by SARS-CoV-2, nor detection of environmental contamination by the virus are yet simple. Complicated techniques of molecular amplification, requiring serious infrastructure and biosafety procedures, limit people everywhere from knowing whether the virus is really in their environment or not. 

The 'Do-It-Together SARS CoV-2 Detective' project, thanks to the support of JOGL and its international team of collaborators, has developed a strategy inspired by techniques used for the ‘GMO Detective’ method, in order to detect the virus causing Covid-19, Corona Detective. Done not only without complicated equipment but with a simple +/- readout, these #CoronaDetective reactions are very specific. Furthermore, controls to ensure sensitive detection, without false positives or negatives, are intrinsic to the surveillance solution. The final product, strips of tubes (or 96 well plates) with dry reagents, could be readily supplied anywhere, without cold-chain dependence; and monitoring tests could be run by ordinary people, without any background in medicine or biology, just some ability to follow simple instructions and common sense.

Human or clinical samples should only be run in settings with access to appropriate biosafety facilities, of course. This project is in full compliance with the OpenCovid-19 Initiative's Biosafety and Biosecurity Guidelines, and is fortunate to include appropriate labs for all levels of validation.

Using openly available information, we optimised components and have defined methods for anyone to use the Corona Detective.

Here are the protocols for making the Corona Detective tubes and for using them (another update to come soon! and don't worry you can view it all, even if you don't make a user account in protocols.io):

https://www.protocols.io/view/freeze-drying-lyophilization-and-manufacturing-of-bpv4mn8w

https://www.protocols.io/view/corona-detective-user-protocol-v2-0-bpwzmpf6

We were very happy to get more support from the 5th round of JOGL microgrants for these open science efforts. The completed JOGL application (Project application pdf from 21mar21) is found in the 'Documents' section of this project page.

Since our last round of JOGL funding, we have submitted a manuscript about the Corona Detective method to an academic journal (Journal of Biomolecular Techniques), and collaborated on an article already available as a preprint and published by peer review now, which reports data with the reactions that include influenza B detection, in parallel with a cell RNA and the N gene target of the SARS-CoV-2 virus. Along with another publication on flu multiplexing with SARS-CoV-2 detection - we should be ready for the flu season down under? :)  

(lol, as thx to the pandemic, there has barely been a flu season recently anywhere...)

We really appreciate getting more support from JOGL, in order to make more reaction tubes (also in Switzerland!) that can be sent to collaborators for further clinical validation, and to continue bioinformatic efforts to 1) make sure important viral variants do not escape detection with our primer set and 2) collect the validation data from international colleagues.

Your help is very welcome! -> and we thank everyone for the open science review, unique (for now) and precious, allowing a community lab like Hackuarium to access top biomol reagents... =) 

See ''needs'' and also "news" for updates, too, especially if you have ideas for at scale production and implementation of our Corona Detective! 

(For surveillance screening and especially in resource-strapped settings, progress is being made, but negotiations about production, licensing fees and use case scenarios in various countries could require efforts of people with all sorts of expertise. Like you?)

We have developed the 'Do-It-Together SARS CoV-2 Detective' project (working towards a #CoronaDetective kit) in order to more readily detect the virus causing Covid-19. This method needs no complicated equipment and has a simple +/- readout, yet is very specific. Furthermore, controls to ensure sensitive detection, without false positives or negatives, are intrinsic to the method. The final product, strips of tubes with dry reagents (or 96-well plates), could be readily supplied anywhere, without cold-chain dependence; and monitoring tests could be run by ordinary people, without any background in medicine or biology, just some ability to follow simple instructions and common sense. We have successfully 'multiplexed' fluorescent detection, for a viral and a control target, using an inexpensive detector.

Open participatory research, together, can help solve today's problems!

You can join us via this Slack channel:

#proj-nucleic-acid-amplification

of the JOGL Open Covid-19 Initiative

(on-boarding for additional participating partners can start here)

Neither diagnosis of cases of SARS-CoV-2 nor detection of environmental contamination by the virus are yet simple. Complicated techniques of molecular amplification, requiring serious infrastructure and biosafety procedures, limit people everywhere from knowing whether the virus is really in their environment or not. We hope to change this.

We not only got good multiplex results for detection of the viral target (N gene) and an internal control (RNAse P), but the reaction with flu primers was also successful, and good progress was already made for further R&D, sample preparations, production and quality control of freeze-dried reaction sets.

Thanks to JOGL's funding for helping us get molecular biology reagents for Corona Detective (especially at Swiss prices!)!

Now we are doing more to produce and distribute reaction tubes to collaborators and gather clinical validation data, while continuing to investigate viral variant populations to make sure our primer set will still detect current infections.

(see complete application in Documents, below)

Conclusions and broad dissemination of the open results

We are documenting the project for dissemination through open-source platforms, so anyone could benefit from these results and replicate the system. 

Here again are the protocols for making the Corona Detective tubes and for using them!

https://dx.doi.org/10.17504/protocols.io.bk44kyyw 

https://dx.doi.org/10.17504/protocols.io.bk43kyyn

The short communication paper to JBT, in press, now, along with a big review that we have also contributed to as part of the 'glamper' academic collaborations, may come out in a special issue! 

Watch this space!

Let us know if you have any questions...

References

1. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28: E63.

2. Nagamine K, Hase T, Notomi T. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes. 2002;16: 223–229.

3. Becherer L, Borst N, Bakheit M, Frischmann S, Zengerle R, von Stetten F. Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection. Anal Methods. 2020;12: 717–746.

4. Wong Y-P, Othman S, Lau Y-L, Radu S, Chee H-Y. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms. J Appl Microbiol. 2018;124: 626–643.

5. Vienna Biocenter's RT-LAMP site. Full of great information, the researchers, who put together this site, had one of the most impressive preprints around this method in the 1st year of the pandemic, but they do not use freeze drying or QUASR detection strategies, like Corona Detective.

6. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009;15: 62–69.

7. Ball CS, Light YK, Koh C-Y, Wheeler SS, Coffey LL, Meagher RJ. Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses. Anal Chem. 2016;88: 3562–3568.

8. Priye A, Bird SW, Light YK, Ball CS, Negrete OA, Meagher RJ. A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses. Sci Rep. 2017;7: 44778.

9.Rabe B, Cepko C. SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification. Proceedings of the National Academy of Sciences Sep 2020, 117 (39) 24450-24458

DOI: 10.1073/pnas.2011221117

10. El-Tholotha M, Bau HH, Song J. A Single and Two-Stage, Closed-Tube, Molecular Test for the 2019 Novel Coronavirus (COVID-19) at Home, Clinic, and Points of Entry. 2020. doi:10.26434/chemrxiv.11860137.v1.

11. Nie K1, Qi SX, Zhang Y, Luo L, Xie Y, Yang MJ, Zhang Y, Li J, Shen H, Li Q, Ma XJ. Evaluation of a direct reverse transcription loop-mediated isothermal amplification method without RNA extraction for the detection of human enterovirus 71 subgenotype C4 in nasopharyngeal swab specimens. 2012. doi: 10.1371/journal.pone.0052486.

12. Segawa T1, Kobayashi Y1, Sase Y1, Itou T2, Suzuki M3, Endoh T4, Nakanishi T1, Sakai T1. Easy-to-use rapid gene amplification method for direct detection of RNA and DNA viruses in sera and feces from various animals. 2014. doi: 10.1016/j.jviromet.2014.01.019.

Not only were multiplex Corona Detective reactions were developed, which work very well to detect viral (NM) and internal control (RP) targets, but also the reaction with flu primers is sensitive and specific (Figure 6C). This means that specific detection Covid-19 is not only possible, but Corona Detective could be used for surveillance testing even in populations potentially co-infected with influenza. Corona Detective results with the XPRIZE semi-final samples were extremely specific, though not sensitive enough to move forward to the finals. For Corona Detective XPRIZE samples, actually, only the 'direct' sample treatment method was really valid, as the sample set sent to Sri Lanka had thawed out before its reception, and it was this team who had done our Corona Detective tests with concentrated samples. Nonetheless, 1-5 copies per microliter of reaction was specifically detected, and a whole set of other viral samples was not detected, another satisfying specificity result. Furthermore, Guy has now increased input volumes of the 'directly' treated samples, to at least double sensitivity (as calculated from volume difference, 2.5x). Further R&D using sets of control RNA targets (18S and 16S RNAs), and another viral RNA set (Lamb) were not so effective as our current reaction sets, however did help us to confirm viral detection in a sewage sample from the EPFL ENAC lab, with ready detection even after 100-fold dilution of the sample. Finally, in the 8-tube format for the multiplex, as highlighted in the past, 5 unknown samples can be tested, much better than just 2 unknowns per 8 tube set, as in the GMO Detective. (In the 96 well format, which can also be produced on the system in Paris, this means 93 unknowns could be tested - a boon for scaling up the procedure, once validation is complete.) We are working towards a surveillance testing use scenario, potentially with families doing weekly tests, and documenting their results with images taken of tubes in the GMO Detective Detector, as also used for our manuscript submission. 

However, also in a 96-well plate and just on a simple trans-illuminator, the endpoint results are clearly yes or no! (as seen also in news items about our XPRIZE semi-final results from last fall...)

One glitch for the project, after promising discussion with a CEO in the US in regards to production at scale, including a zoom session the day of the capital insurrection (! there was a historic American flag behind him, too, in the call), completed MNDAs, and detailed information shared, no further communication has been obtained. We are new to all these sorts of negotiations, and are trying to chalk it up as a learning experience. Nonetheless, we feel optimistic about the progress with JOGL's FITZ initiative, and wonder if it will help us get past regulatory hurdles to bring this project to production and real use in surveillance testing.

It must be noted, in addition, the glass-milk method for concentration of samples tested (as in reference 9 above) was not so reproducible in our hands as other, more costly, RNA extraction kits, which were what was used by our Sri Lankan colleagues with their XPRIZE samples, it should also be noted. Indeed, already for our XPRIZE samples, Guy gave up on the glass milk method and stuck with the 'direct' results. Therefore, in the Hackuarium lab, TCEP was instead ordered (not the glass milk reagents, as in the proposal submitted last fall). The ambient temperature stability of the 100xTCEP solution and ease of use, are key improvements. Currently, we very much look forward to reception of an actual control virion preparation (inactivated and only requiring BSL1 facilities, but of course) from BEI Resources, in order to do further R&D work in this regard, while also exploring possibilities for local tube production with a new team member.

Abbreviated Project Timeline

·     producing and shipping 1000s more Corona Detective multiplex and 'single-plex' tubes to international colleagues. 

·   continuing bioinformatic efforts to:

   1) make sure important viral variants do not escape detection with our primer set and

   2) collect the validation data from international colleagues.

         In Ghana, there are already plans for this validation work to enable their official application to local regulatory   agencies to allow authorised use. Collaborators in Cameroon, Sri Lanka and Chile are also running validation tests on Corona Detective reaction tubes, with clinical samples according to local regulations.

These timeline items can all be fulfilled in weeks (once orders are obtained, to maximum two to three months for analysis of the validation results), after orders are in hand for more production (FR) and initial production tests (CH), with a new member of the team (Clément Bordier, who is not only interested in the project, but has a lyophiliser in an ISO CE lab, not so far from the Hackuarium lab).

Again, our long term aim is to progress to the point where Corona Detective actually being used for surveillance testing, as about 40% of infections may derive from asymptomatic carriers...

(If vaccination could get going faster, maybe we will be happy to use this experience to help out for a future pandemic, but if people don't stop giving the virus the opportunity to outwit current vaccines, maybe community surveillance will remain an essential tool to combat the pandemic, for some time to come...) 

We all know already about problems testing for the virus that causes Covid-19. Everyone is a stakeholder in the Open Covid-19 Initiative, and this project.

Here are some further proposal application points:

Will you need assistance with the regulation system? if not which Regulatory system do you plan on distributing the product? Please elaborate (please see: Regulatory-Strategies)

At least at first, this project will be either WeProvideNonMedicalDevices-Public or WeProvideInstructions: and it will be for research use only, for viral detection, not a medical diagnostic.  

To get Corona Detective reaction tubes used is already half-way there, in terms of the regulatory landscape, as the viral primer set we use, NM is already FDA Approved.

(Link to EUA announcement: https://www.fda.gov/media/139937/download )

Have you talked to medical staff about the feasibility of your project? What did they say?  

Enthusiasm was obvious when we talked to others, including medical professionals, about this project. One German doctor from the Open Covid-19 Initiative already asked about possibilities for clinical tests in Dortmund with this proposed solution to the testing problem.

What impact do you feel your product could have?

The impact of this project will potentially be very high, as molecular detection tools for the virus causing Covid-19 are only available in specialised laboratory settings now. The ‘DIT SARS CoV-2 Detective’ solution could allow average people to test for the presence of virus, wherever they might be. 

What do you think would make your project a success?

Getting the #CoronaDetective kit out to the world after collaborative experiments for parallel tests and validation by multiple partners in different countries involved in this open participatory research and development work would really make this project a success.

Please list the known issues, potential risks, grey-areas, etc in your project:

**Making sure people know to never open kit tubes after the reaction has run, particularly ones that gave a positive result, is the biggest issue around using this method. The worst would be if samples got contaminated by end-product. If such contamination occurs, people might panic, believing there is much more virus around than there really may be. This risk is mitigated, however, by the requirement to always have a negative control for each reaction set. If the negative control shows up positive, cleaning well with diluted bleach and alcohol before running any new set of tests is essential.  

*Another issue is the fact that all the molecular reagents, in particular the enzymes needed, must be purchased from biotech companies like New England Biolabs. In the Open Covid-19 Initiative, however, a group (FreeGenes) is working towards open alternatives. Even in the short term, we hope that the companies might be convinced to help make even the initial #CoronaDetective kit a feasible option.

What steps have you taken to ensure your solution’s safety? How advanced are you in this process (if applicable)? Please check the Biosafety and Biosecurity guideline of OpenCovid19

Risk assessment has been reviewed. For most proposed tests no live virus would be utilised, with only parts of genes either as DNA or RNA used as the positive controls, except when a partner has access to a BSL3 lab.

Have you planned your manufacturing process to ensure quality, what are the steps you have taken? How advanced are you in this (if applicable)?

Dependence on molecular biology companies for primers and reagents means there is some guarantee applicable.

Furthermore, the robotic/manufacturing pipeline of one partner (FR) has already been validated for the GMO Detective kit, and other projects.

Project partners are strongly committed to the principals of open science and participatory research, and have a long-term investment in showing that 'Do-It-Together' principles are the way forward, in order for our modern world to resolve problems. While several open public lab partners still strive to become sustainable, their implication in projects like these (and many more), means making all this work well is an important priority. To increase scientific literacy of the public, through use of such tools, adds to the stakes, as this is one of most partners' key aims.

While we wrote this in the last two applications: Scalability is already ensured, as we have access (as long as Covid-19 lockdown is not too strict) to in-house manufacturing capabilities, using automation to allow creation of ~500 freeze-dried kit strips per day. (FR) - In fact we now realise that scalability is still a huge challenge ahead. More support will facilitate matters, but more is also required. (Maybe FITZ will help get this to happen?) 

This project will develop a completely open, shareable and transparent method.

Social media tools, websites, wikis, working groups, regular media, and the all important 'word of mouth' will all be utilised to help communicate progress and disseminate results of this project. (For instance, here are a just a few elements of our current web presence: www.hackuarium.ch, wiki.hackuarium.ch https://gmodetective.com/, https://openfiesta.space/ www.genomicintegrity.org  

Twitter: @hackuarium, @AGIRgenomes  

Facebook: @hackuarium, @genomicintegrity.org)

Lead Team experience:

Guy Aidelberg developed the GMO Detective assay and has worked on similar systems for ZIKAV/DENGV and Rachel Aronoff, Fran Quero and Ali Bektas have worked with this method for various projects.

The project involves multiple international laboratories (already from 6 different countries):

Guy Aidelberg and Fran Quero are in Paris at the CRI (FR), Rachel Aronoff is in Lausanne working with the open public lab Hackuarium (CH). Dr Samuel Sakyi and Harry Akligoh are from Ghana’s Kwame Nkrumah University of Science & Technology and Kumasi Hive labs. Thomas Mboa, Stephane Fadanka and Nadine Mowoh are in Cameroon with the open science MboaLabs (CM). Fernan Federici is in Chile, with his own academic lab (CL), and finally there are several partners in the United States (USA): Ali Bektas at UC Berkeley in California, Chris Monaco at the CDC in Atlanta, Ellen Jorgenson with the company Aanikabio in NY, and Sarah Ware, founder of two independent labs in the Chicago area: BioBlaze Community Bio Lab and Lizzy Blossom Ag Services.

JOGL awarded initial funding to the project in the first round of JOGL microgrants (April 2020), and in the 4th round of microgrants (October 2020). Hackuarium provided a small microgrant, and the work in Paris has been primarily funded by Guy's grants and Ariel Lindner's lab in the CRI (where Guy just finished his PhD - congratulations!).

The production work in Paris essentially costs about 3000 USD just for ordering all the components for the Corona Detective tubes - and ultimately costs about 2 USD per reaction - so for 1000 tubes, 2000USD is really needed on that end. Costs

For the further R&D in Switzerland, the inactivated viral control was already ordered from BEI resources, to work on the sample preparation side of things (with the TCEP-EDTA solution and heating, shown to work well) for a user friendly protocol. With the Bordier lab, we are looking into the lyophilisation procedure further, while Guy and Fran plan to continue to make and distribute reaction tubes to international collaborators according to the established protocol. Fran also has more plans for working on the open enzymes and open hardware for reaction incubations.

To help us out further, for production and additional R&D costs, 4000 euros from JOGL in this round would be really great to obtain.

• for the French team at CRI, 3000 euros for more significant contribution to tube production runs
• for the Swiss team at Hackuarium, 1000 euros for more enyzmes and molecular biology consumables (filter tips, etc)

In the Documents section of this page, you can also find:

1) an 'infographic' style image from Ref 7, explaining how the fluorescence quenching of the Quasr method works, and

2) a generic qPCR result, from previous work (CH), showing how, upon amplification, signal can be seen to increase over time.

3) a preliminary budget document summarising most initial needs for project partners.

4&5) the complete application with budget details, our 2nd request for JOGL funding (18oct2020, with clickable links in the second version) .

6&7) 'archived' pdfs of the last version of this project page, and its news items!

8) the complete application with budget details, now our 3rd request for JOGL funding (20mar21)

Section 8:

JOGL contributions really kick-started the R&D on this project at Hackuarium and for the whole Corona Detective team, and we are all extremely grateful for their hosting this collaboration!

None of this work could be possible without the generous support of JOGL and the entire open community, which has brought us all together and supported us every step of the way, during this tumultuous year! (For further budget details see Document section.)

Our new Corona Detective Logo in the Project Banner spot now (20mar21), is just a hack, not the proper svg yet, and any feedback is welcome!