Do-It-Together SARS CoV-2 Detective

Do-It-Together SARS CoV-2 Detective

#CoronaDetective

A project to develop an open-source detection method for SARS CoV-2, analogous to the GMO Detective.

Created on: April 03, 2020

by Francisco Javier Quero Lombardero, Guy Aidelberg, Rachel Aronoff

Participating to challenge(s): Covid19 Diagnostic and Detection

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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 will develop 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 predicted to be 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, targeted regions of the virus have already been analysed, and one even shown to be identifiable by team members with such molecular techniques. Now we aim to optimise components and to make a validated method for anyone to use.

Your help is welcome!

Elevator pitch / Abstract

We hope to develop 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 are gearing up to try for 'multiplexed' fluorescent detection, using an inexpensive (even DIY) detector, but with a partner project (Slack #proj-neb-lamp-test), we would also be glad to find out whether a simpler color change reaction will be sufficient for valid results.

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

How to contribute


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)

Problem Statement

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.

Objectives & Methodology

The project proposal is based upon ‘loop-mediated isothermal amplification’(1,2), or LAMP, a molecular detection strategy that allows identification of specific nucleic acids in samples, with the possibility for colorimetric or fluorescent visualization of results. The reaction uses 4-6 primers specific for short target regions (usually less than 300nt) and is run at a single temperature (about 65 degrees Celsius), meaning no special thermal cycler or even incubator is required. The reaction also completes in a short time (15-45min, depending on the target region)(3-6). Furthermore a special quenched fluorescence system (QUASR-rtLAMP) allows binary, "yes/no" detection of specific nucleic acid sequences and even multiplexed reactions (7,8). In summary, such loop-mediated isothermal amplification (LAMP) assays offer several advantages over qRT-PCR, the current standard for SARS CoV-2 laboratory diagnostics, as it is relatively inexpensive, does not necessarily require nucleic acid purification prior to assay, and provides a simple visual or fluorescent detection of positive samples. The proposed solution would consist ultimately of 8-tube strip ‘test kits’ containing lyophilised components (primers and enzyme) and including controls, so that they could be sent anywhere by post for validated detection of SARS CoV-2.


Parallel experiments by international partner laboratories from 6 countries (FR, ES, CH, USA, CM, CL) will ensure reliable development of the proposed #CoronaDetective kit.


Objective 1: Develop a functional Isothermal Amplification system, robust enough to detect SARS CoV-2 in samples. 

Loop-mediated isothermal amplification can amplify a few copies of a nucleic acid target to 10^9 copies in less than one hour, even when large amounts of non-target DNA are present or other biological substances. RT-LAMP has been shown to be sensitive and specific enough to detect RNA viruses from biological samples such as blood, serum, urine, cerebrospinal fluid, tissue, and nasal swabs, without needing purification of RNA (12). We will test several openly available primer sets against SARS CoV-2 gene sequences and a control human gene to obtain optimal components for the #CoronaDetective kit. Most lab tests in the context of this proposal will evaluate reactions only with positive control DNA, but other partner’s lab access means we can also validate our most efficient component mix with real viral samples. 


Objective 2: Develop a kit that can be sent without depending on cold chain maintenance.

Lyophilised components driving LAMP reactions are surprisingly stable, and one partner has access to high-quality robotic tools and freeze-drying equipment, allowing large-scale production of the kit, based around 8-tube strips. These would be mailed off to partners around the world for tests, documentation and comparison of results. 


Methodology (technical details)


(Day0) Reception of reagents (primers, enzymes, buffers, positive control DNA/RNA)

Primers would be ordered and obtained as lyophilised powder. Commercial enzymes and buffers and positive control plasmid (2019-nCoV_N_Positive Control plasmid from Integrated DNA Technologies) will be used, although there are hopes that open alternatives may become available. Two primer sets from the N gene region are initially proposed for this project, one from Zhang et al (9) and the other from the ‘Mammoth protocol.’ Additionally, a promising set of LAMP primers for the SARS CoV-2 Orf1ab gene (5), will be tested. Finally, a control primer set for the mRNA of the human housekeeping gene is proposed. These primers would be specific for mRNA, and can even be useful as an internal human RNA control for multiplex LAMP applications.


More details for how to design primers for the Quasr detection can be found here and here (in particular for these targets in the SARS CoV-2 N gene). Here is the open notebook (Benchling) page pulling details together.


(Days 1-14) First tests and primer set optimisation

Complete details for running basic isothermal amplification protocols can be found here


Optimisation is performed by varying ratios of primer pairs in each set, and seeing which condition gives the fastest amplification time. Optimised primer concentrations are then titrated with varying amounts of MgSO4 to determine the best concentration for each primer set. Use of qPCR machines will facilitate the empirical detection of these optima. (CH, ES)

In parallel, viral RNAs can be tested at the CDC facilities with BSL3 conditions, to make sure the conditions are also suitable for such samples. (USA)


(By day 28) Validation of sensitivity and robust detection capacity

Dilution series with the positive control plasmid sequence (or maybe synthetic RNAs and hopefully viral samples at the CDC BSL3 lab) will allow determination of the limit of detection for the reactions. 


If the colorimetric detection is sensitive and reliable enough, as being tested in the partner project #neb-lamp-test, the next section and other phrases in italics (below) can be omitted.


(From day 15) Labelled primers and quenchers arrive for the best N set of primers

For the labelling of one set of N primers, it looks like a fluorescein molecule at the 5’ end of the forward internal primer (FAM-FIP) with a Quencher at the 3’ end of a very short (10 bases) complementary sequence looks very promising, but awaits the empirical tests above. 

To validate the best conditions for these labeled sets, a SYTO dye to detect in a different channel from fluorescein (i.e. ROX) will allow test reactions to be assessed in a qPCR system. Partners can also score the fluorescence of the results with the ‘GMO detective’ fluorescence detector tube holder.


If all looks well, tests of multiplexing will also be possible (with all 14 primers put together), which would require another (i.e. TRITC) label on the control gene set. Multiplexing would have the advantage that each set of 8 tubes could be used to test for 4 or 5 samples, rather than only 2 samples, as in the GMO Detective. With multiplexing, both positive results in one tube give yellow fluorescence. If one more control is desired for the combination, 4 samples could be tested from one strip (i.e tube 1, negative control; tube 2, mRNA + control, red; tube 3 N + control, green, tube 4 both + controls, then tubes 5-8 for samples). Feedback of end-users will be important to decide this detail.


(Day 30) Large-scale orders of best sets for lyophilisation (to be put together in FR) into the 8 tube strip format

Will need to get special approval to go into the lab to use the equipment there (FR), if France is still under lockdown.


Validation of the lyophilised ‘kits’ for detection

Mail tube strips to all partners (CH, ES, FR, USA, CL and CM) for tests, scoring fluorescence with the GMO detective rig and collecting all results on an open platform.


(Before day 60) Conclusions and broad dissemination of the open results

We will perform the final documentation of the project and its dissemination through open-source platforms, so anyone could benefit from these results and replicate the system.

State of the art

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.

Progress report

A great deal of information about the virus was already available openly, and close examination of available sequences and preprints (refs 5,9,10) has confirmed several sets of promising primers to detect conserved COVID-19 viral targets. Several primer sets that look like they would be suitable for the Quasr method, and one set has already been subjected to initial tests in the partner project #proj-neb-lamp-test. Optimisation of reactions and determination of the best primer sets for inclusion in the #CoronaDetective kit are the key project aims, once funding enables us to get necessary reagents to project participants. 


Abbreviated Project Timeline

  • Reception of reagents (primers, enzymes, buffers, positive control DNA/RNA) (Day 0)
  • First tests and primer set optimisation (Day 1-14)
  • Labelled primers and quenchers arrive to test also (from Day 15)
  • Validation of sensitivity and robust detection capacity (by Day 28)
  • Large-scale orders of best sets for lyophilisation (in Paris and?) into the 8 tube strip format 
  • Validation of the lyophilised ‘kits’ for detection (CH, ES, FR, USA, CL and CM)
  • Conclusions and broad dissemination of the open info (before 2 months)

Stakeholders

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. We do not initially plan on going for FDA approval or similar regulatory agencies as many companies are already doing that see:https://www.finddx.org/covid-19/pipeline/


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.

Impact strategy

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.

Ethical considerations

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 initiated. 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.


Have you planned the testing, verification and validation of your solution? How advanced are you? (if applicable)

As the partner project #proj-neb-lamp-test has already shown viral sequences (as control plasmid DNA) were detected with one set of primers, and even already improved the limit of detection by a change in ratio of the primer set (doubling the so-called ‘loop’ primers), we are confident that within two months of work, we should already have an initial ‘test kit’ to validate, based on the most robust and sensitive primer sets for the virus and control gene sequences.

Sustainability and scalability

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)

Communication and dissemination strategy

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.

Funding

A 500chf minigrant from Hackuarium has already been approved for this project (CH).

There is also at least 1000euro available from another viral project (FR)


Additional funding is being sought for all partners.


Depending upon whether biotech colleagues can be convinced about providing special prices for these efforts, each participating lab would likely need to be funded for:

x3-4 sets of primers (150-200) and possibly

x2 sets of labelled primers for Quasr detection(400),

+ enzymes/buffers/nucleotides (500),

and at least Proteinase K and DnaseI (150)

for a total of around 1250euros per lab. 


Adding consumables like tips and tubes, especially for the non-academic labs, this can be very important, particularly in this context, could add another 100-200 or so. If more tests with RNA controls alone are essential, about 200 would be added to this total.

Thus, initially, this project would hope to obtain about 1800 each for two laboratories (ES and CH).

(If special solutions for sampling are included, this could add even more.)



For more information and detailed budgets, please see this spreadsheet.

The completed application template document (with more details) is also available.


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 CH and ES partners (phone calls will be made!)




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#CoronaDetective_Budget_start partnerRA_CH.pdf

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