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 similar to that used for the ‘GMO Detective’ method, in order to detect the virus causing Covid-19. Done not only without complicated equipment but with a simple +/- readout, the #CoronaDetective is very specific. Furthermore, controls to ensure sensitive detection, without false positives or negatives, are intrinsic to this solution. The final product, strips of tubes 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:

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

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

We are hoping to get more support from the 4th round of JOGL microgrants for these open science efforts, and look forward to demonstrating how well the method works in the XPRIZE semi-finals! The completed JOGL application (JOGL2_CoronaDetective_funding from 18oct2020) can be found in the 'Documents' section of this project page.

Your help is very welcome! -> also for the open science review...  :) 

See ''needs'' and also "news" for updates! 

In brief: we hope that 1) the XPRIZE 'blinded proficiency test' goes smoothly, 2) 1000s of tubes will be sent to collaborators for further clinical validation, and 3) we can develop a triplex version of Corona Detective, to include flu virus detection.

We are developing the 'Do-It-Together SARS CoV-2 Detective' (or #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 predicted to be 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, 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 got good multiplex results for detection of the viral target (N gene) and an internal control (RNAse P), and Guy is working on the sample preparations, and also doing lyophilisations for shipping to partners, especially SLINKA, that in Sri Lanka, along with Guy in Paris, will be testing the XPRIZE 'blinded proficiency' test samples.

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

Now we want to send thousands of tubes to collaborators and try for 'triplex' detection, including a flu target.

(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 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

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. Yu L, Wu S, Hao X, Li X, Liu X, Ye S, et al. Rapid colorimetric detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform: iLACO. medRxiv. 2020; 2020.02.20.20025874.

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. Zhang Y, Odiwuor N, Xiong J, Sun L, Nyaruaba RO, Wei H, et al. Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv. 2020; 2020.02.26.20028373.

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.

Multiplex Corona Detective reactions were developed, which work very well to detect viral (NM) and internal control (RP) targets. This means that very sensitive and specific detection is possible. Furthermore, in the 8-tube format for the multiplex, 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.)

Abbreviated Project Timeline

·     producing and shipping 1000s of Corona Detective multiplex tubes to international colleagues.

·     ordering the flu primer set and optimising reaction conditions (relative primer concentrations and magnesium levels).

·     testing these primers in Corona Detective reactions, in combination with the usual multiplex components, and alternatively with the other control RNA primer sets.

·      analysing results of these validation attempts, gathered with the simple app from our international colleagues.

These timeline items can all be fulfilled in weeks (to maximum two to three months for analysis of the validation results), after orders are in hand for production (FR) and triplex (CH) tests.

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.  

(at least initially - if we get through to finals of the XPRIZE, it may well be that their validation will allow more use-cases.)

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. However, 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.

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)

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 is in Paris at the CRI (FR), Ali Bektas is at UC Berkeley in California, Rachel Aronoff is in Lausanne working with the open public lab Hackuarium (CH), and Fran Quero is in the midst of masters studies in Shenzhen, China, but is now in Madrid with access to lab equipment at the Carlos III Hospital (ES). 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 3 partners in the United States (USA): Chris Monaco at the CDC in Atlanta, Ellen Jorgenson with the company Aanikabio in NY, Sarah Ware, founder of two independent labs in the Chicago area: BioBlaze Community Bio Lab and Lizzy Blossom Ag Services, and Isabella Zorra also working with the BioBlaze Community BioLab.

JOGL awarded initial funding to the project in the first round of JOGL microgrants (April 2020). Hackuarium provided a small microgrant: and the work in Paris has been entirely funded by Guy's grants and Ariel Lindner's lab in the CRI (where Guy is currently finishing his PhD), to date.

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.

For the further R&D in Switzerland, the cost of a primer set, with one primer tagged fluorescently (Texas Red is a likely choice, to go with the Fam and Hex targets) and a complementary quencher for the QUASR detection costs at least 250 euros for the tag and quencher and about 70 euros for the rest of the primers (i.e. for the Flu target), while Hackuarium has already spent almost 4000 chf for all the various enzymes, nucleotides, etc. (and should include costs for sample extraction).

To help us out further, for production and additional R&D costs, 700 euros each, for 1400euros total from JOGL in this round, would thus be really great to obtain.

• for Paris team at CRI for some contribution to tube production
• for Swiss team at Hackuarium for primers, and sample extraction reagents (glass milk and NaI)

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) .

Section 8:

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