Using DIYBio tools to improve RT-LAMP Protocols

Using DIYBio tools to improve RT-LAMP Protocols

#DIYBiovsCovid19

A project to test DIY hardware which could enhance quality, user-friendliness, reliability, and reproducibility of RT-LAMP protocols.

Created on: April 15, 2020

by Antonio Lamb

Participating to challenge(s): Evaluation and Validation of Open Source Solutions

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Introduction 


Problem and Background

Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) is a very promising and established nucleic acid amplification technique that can be used for rapidly detecting SARS-COV2, the virus responsible for COVID19 (1). Various research groups around the world are working on optimizing this test for widespread use (1–3). The most exciting aspect is that the test materials can be freeze-dried, which would remove the need for cold storage. With some optimizations, it could be widely deployed to areas which have no refrigeration (4). There are already several groups in the OpenCovid19 Initiative who are working on optimizing RT-LAMP protocols and making the procedure as easy and widely accessible as possible. The Do-It-Together SARS-COV2 Detective (led by Francisco Lombardero, Guy Aidelberg, and Rachel Aronoff) is working to develop a freeze-dried reaction kit that would only require the addition of the test sample and water. One of the only hardware requirements is a source of constant heat, from either a heat block or a warm water bath. But how sensitive is the assay to minor fluctuations in temperature? Could an inexpensive temperature-controllable DIY heatblock remove some variability and further improve the test’s accuracy?


Solution summary in simple terms

We propose running a simple RT-LAMP experiment comparing the efficacy of the best RT-LAMP reaction conditions validated by the diagnostics teams under several temperatures using Gaudilabs PocketPCR, a conventional PCR machine, a heat block, a water bath, and a cup of hot water. 



Solution summary in technical terms

Although RT-LAMP is a very robust reaction and can tolerate a fairly wide range of amplification temperatures, there are reports that variations in temperature do have an effect on the accuracy of the assay. In an RT-LAMP experiment conducted by Lu, et. al. to amplify a fragment of the Cucurbit chlorotic yellow virus, they found that sub-optimal temperature ranges produced low concentrations of amplification products (5). Fluctuations in temperature due to the lack of controlled heating and cooling may have a negative effect on product amplification. Heat blocks cost anywhere between $40 to $250. GaudiLabs open source PocketPCR, which also functions as a programmable Polymerase Chain Reaction (PCR) thermocycler, seems like a promising device to deploy since it can also be used for an array of advanced PCR-based assays and cloning projects. It has internal temperature controls which allow for the precise adjustment of reaction temperatures, and is extremely user-friendly and easy to build.




State of advancement of the project

Currently, we recruited a team of 8 volunteers, 4 of which have advanced degrees in molecular biology and computer science. We have also secured a makerspace in Basel (the Starship Factory), where we have access to 3D printers, laser cutters, and soldering tools. We also have a mentor and advisor from the University of Basel (Dr. Timm Maier), who will provide guidance to the group. We are currently building a community lab here to further assist with optimization of the RT LAMP detection assay. Securing a small microgrant would allow us to get started much sooner. We have an excellent team, an experimental plan, and a project timeline. We have already built some of the core equipment, such as the PocketPCR. We have also joined forces with all of the DIY diagnostics teams on JOGL, and we have created a page for the project. The image below summarizes the current state of advancement of the project:



Project Timeline


  • Phase I - Purchase RT-LAMP reagents from NEB and/or OptiGene, a water bath, heat block, PCR machine, oligos, and control samples (1-2 weeks).
  • Phase II - Conduct a controlled experiment which tests the effects of temperature consistency on template amplification using the various heat sources.
  • Future Plans - Once the rest of the DIYLab is operational, begin supporting basic cloning, protein expression, and purification activities of the OpenCovid19 Initiative’s molecular biology research groups as needed.


Project Implementation


In the short term, our project solution will increase the number of trained researchers and scientists working on the Open Covid19 Initiative’s RT-LAMP diagnostic, and will help determine whether the GaudiLabs PocketPCR can be used with the RT-LAMP assay. In combination with parallel crowdfunding efforts, this project will also result in 6 trained DIYBio research staff coming online in Basel, Switzerland to help with enzyme purification, molecular cloning, and nucleic acid amplification testing. In the long term, if our proposed engagement model and project campaign are successful, it will inspire more DIYBio groups to join the OpenCovid19 Initiative, and may serve as a model for how we can engage the community in the future. We want to see as many DIYBio labs join us as possible from around the world. The sun should never set on the OpenCovid19 Initiative. 


Other potential collaborations of interest we would like to explore once we have established a community lab in Basel include the following:


  1. The Enzyme Production Group
  2. We propose providing assistance in cloning and plasmid assembly to help them express their histidine-tagged enzymes, as well as several other expression cassettes.
  3. We can also assist with feasibility assessments to explore whether these purification protocols are user-friendly and can be performed outside of a normal laboratory setting.
  4. The Bioreactor Group 
  5. We propose exploring new strains of halophilic bacteria (Halomonadaceae) as new host systems for recombinant plasmid and protein production. These systems would be beneficial for widespread use because they do not require sterile growth conditions and could therefore be used in a variety of settings where sterile cell culture is not possible.
  6. The Nucleic Acid Amplification Group
  7. We have requested samples of freeze-dried RT-LAMP reaction kits and would like to assist with identifying a suitable indicator dye or other detection reagent which is stable when freeze-dried, or can be added to the reaction after it is reconstituted in water.


Our long-term objective is to successfully build a low-cost, community-run laboratory in Basel, Switzerland so that we can provide additional support for the existing OpenCovid19 molecular biology projects. Our project serves as a model for how small, low-cost microgrants could be quickly and efficiently deployed to answer basic research questions.


Methodology

Our first experiment (testing RT-LAMP amplification) will be a fairly simple and straightforward experiment. Using approximately 20 or more reactions in each treatment or control group, we will follow the most successful RT-LAMP protocol and primer sets so far characterized by the Diagnostics Groups under normal in-the-field use conditions (cup of heated water) vs. Gaudi’s PocketPCR vs. heated water bath vs. heat block vs. a conventional PCR machine. The result may help us determine whether the PocketPCR device can provide accurate and consistent results that are similar to or better than other methods.


The project will be executed in the following way:


Phase I: Build the PocketPCR (complete). Procure Warmstart RT-LAMP kit from NEB or OptiGene and ancillary reagents (300 euros), PCR machine (500 euros), water bath (120 euros), and heat block (120 euros). Secure all ancillary lab materials (pipettes, etc.) from a parallel crowdfunding campaign. 


Phase II: Execute a controlled experiment which tests the following parameters:


Future Plans:

In addition to sharing the results of this simple experiment, we will document and share all reliable and low-cost sources of reagents and materials we find for the benefit of the OpenCovid19 community while we build our DIYBio lab. We have already successfully assembled the PocketPCR machine, and we have plans to purchase or assemble the remaining material. We will maintain a laboratory notebook that will be accessible for review by other collaborating teams. We will follow basic and reproducible molecular biology techniques to carry out our experiments.


Materials required are listed here: 


A PCR thermocycler (500 euros)

Heated water bath (120 euros)

Heat block (120 euros)

NEB RT LAMP kit or OptiGene RT LAMP kit (300 euros)

Oligos and template (provided by OpenCovid19)


Results/Expected results

At the end of the project, we will make an informed recommendation on whether the PocketPCR provides any improvement in the accuracy of the RT-LAMP reaction, and whether constant temperature really plays a noticeable role in the sensitivity of the assay. We also expect to have an operational molecular biology lab and a team of 8 scientists and volunteers that is ready to assist the diagnostics teams with further projects to optimize a low-cost SARS-COV2 detection kit. 


In the process of building our DIYLab, we will also have several documented sources of low-cost materials and manufacturers which we will share with existing and future labs. We will also assist other potential teams in procuring equipment so that they start actively participating in research activities sooner.


Safety, quality assurance and regulation


Our project carries a minimal level of safety and environmental risk. We have performed a biosafety assessment and determined that all of our proposed work and experiments can be carried out in a BSL-1 laboratory, since we will primarily be working with non-pathogenic bacterial systems and amplifying small synthetic DNA/RNA fragments of the SARS-COV2 virus. Since we are working out of the Starship Factory in Basel, which also shares a space with BASF, we can get assistance in arranging proper waste disposal as necessary. 

We have two trained scientists on the team and we will have a safety training that must be completed before anyone may participate in wet lab work. We also have a PhD scientist from the University of Basel (Dr. Timm Maier) who will provide guidance and mentorship when required, and can help ensure the quality of the work we are doing meets the proper scientific standards. Basic lab safety precautions will be taken, such as wearing gloves and eye/face protection when required. We will maintain a Safety and Security Plan which we will adhere to. We will also have designated a biosafety officer to help implement and ensure adherence to policies. Chemicals and reagents will be stored in a safe manner. Since there is already a community laboratory based on the French side of Switzerland (Hackuarium), we would also be extremely grateful to receive support and safety guidance from them since they already have experience setting up a community lab.

Lastly, limiting the number of individuals working in the lab, social distancing, and proper PPE will be used as necessary to adhere to all local laws regarding the Covid19 quarantine.


Impact, issues and risks


What impact do you feel your product could have?

It is important to know how the RT-LAMP reaction fares not just in tightly controlled laboratory conditions, but also in real-world settings. We need to investigate how important constant temperature is for the reaction. If we do not account for it, our estimations regarding the sensitivity of the test may not be as accurate.


In addition, if we are successful in building a low-cost lab for only a few thousand euros that provides meaningful support to OpenCovid19 R&D, it would provide greater justification to fund additional microgrants for DIYLabs working on specific Covid19 projects. 


What do you think would make your project a success?

Although having proper equipment and funding are important, by far the most crucial part of a successful project is the team. We have a strong, diverse team that is highly motivated and trained to conduct scientific research.


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

There are several potential issues we may run into. We may have challenges getting materials on time, or we may need to troubleshoot an assay if an experiment does not work as planned. In addition, being a community laboratory that must adhere to the local laws and regulations also comes with its own challenges. We intend to work with existing DIYBio labs in the area to ensure that things go smoothly. 


Team Members


Antonio Lamb - Lead Scientist 

Expertise: Project Management, Bioinformatics, Pharmacokinetics, Molecular Pharming, Infection Biology


Education: 

  • B.Sc. Molecular Biology (University of California, Santa Cruz)
  • Post Bacc. Applied Bioinformatics (University of California, San Diego)
  • M. Sc. Molecular Biology (University of Basel) (in progress)


Previous Publications:

Mackenzie-Lamb, Viktor Antonio, Simon Porphy Jegathese, and Kwang-Chul Kwon. (2017). DNA Constructs for Manufacturing Bio-Therapeutic Polypeptides for Use in Animal Vaccines and Therapeutics. WIPO 2017180616. World Intellectual Property Organization, Filed October 20, 2017.


Kwon, K.-C., Lamb, A., Fox, D., & Porphy Jegathese, S. J. (2019). An evaluation of microalgae as a recombinant protein oral delivery platform for fish using green fluorescent protein (GFP). Fish & Shellfish Immunology. https://doi.org/10.1016/j.fsi.2019.01.038


Pat Mächler - Technical Lead

Expertise: Project Management, User-Centered Design, Machine Learning, User Research

Education:

  • B.Sc. Computer Science (University of Basel)
  • M.Sc. Computer Science (University of Basel)
  • MAS Human-Computer Interaction Design (Universities of Rapperswil & Basel)


Claudius Röthlisberger - Machinist

Expertise: Carpentry, 3D printing, electronics


Rishika Agarwal - Scientist and Biosafety Officer

Expertise: Immunology, Cell Biology

Education:

  • B.Tech. Biotechnology (Vellore Institute of Technology, India)
  • M.Sc. Biotechnology (Northwestern University, USA)
  • PhD candidate (University of Basel) (in progress)


Christoff Galvao - Scientist

Expertise: Molecular Biology

Education:

  • B.Sc. Molecular Biology (University of Basel)


Team experience


Antonio Lamb

Antonio is a seasoned Entrepreneur and molecular biologist. He is the CEO & CoFounder of MicroSynbiotiX, an algal synthetic biology startup based in Ireland and is an active member of the DIYBio community, having previously been a volunteer member of BioCurious Labs in California. While working with MicroSynbiotiX, he published a peer-reviewed experimental paper on the pharmacokinetics of plant-based oral vaccines. He and his team also successfully expressed several recombinant subunit vaccines and dsRNAs in microalgae which are currently undergoing animal trials. Antonio now lives in Switzerland. He is a M.Sc. student and an online teaching assistant at the Biozentrum in the University of Basel during the Covid19 lockdown.


Pat Mächler

Pat is a product designer and computer scientist. Pat’s two master thesis related to polynomial algorithms for multi-agent path planning, as well as to the user-oriented, digital organisation of informal groups at the universities of Basel and Rapperswil. Pat currently works as a freelance user researchers and teacher for user-centered design and ICT. Besides Pat is a DIY hobbyist, and thus became the chair of the FabLab Starship Factory. Furthermore Pat has co-founded an international NGO and is its secretary for UN matters, as well as one of its leading social media representatives. 


Claudius Röthlisberger

Claudius is an excellent craftsman. He is a skilled carpenter and hardware hacker who regularly works out of the Starship Factory in Basel, Switzerland. He advises groups who work in the makerspace.


Rishika Agarwal

Rishika worked at a biotech startup in Chicago, USA where she tested immunomodulatory nanoparticles in blood cells and nanoparticles for the topical treatment of psoriasis. She’s currently working in developing skin organoids. Furthermore she’s the president of the Science Slam Club in Basel.


The DIYBio lab will operate out of the Starship Factory, a community makerspace located in Basel, Switzerland, where we have access to 3D printers, soldering tools, and laser cutters, etc. 


Funding 

We would like to ask for a grant of 1040 euros for the following items:

A PCR thermocycler (500 euros)

Heated water bath (120 euros)

Heat block (120 euros)

NEB RT LAMP kit or OptiGene RT LAMP kit (300 euros)

Oligos and template (provided by OpenCovid19)


We are also crowdfunding to raise a total of $3,765.86 (roughly 3500 euros) to open our DIYBio Lab in Basel, which we will raise in parallel with the grant through external crowdfunding. We hope that the grant can be used to promote our crowdfunding campaign, allowing us to secure additional equipment to do more collaborations with the OpenCovid19 molecular biology groups. Currently, we are still in Phase I of our project (securing and building core equipment), however, we have made a significant level of progress. Already, we have 6 team members who are dedicated to seeing this project through, and we have already built the PocketPCR machine and some other core pieces of inventory.


Sources

1. Yu,L., Wu,S., Hao,X., Li,X., Liu,X., Ye,S., Han,H., Dong,X., Li,X., Li,J., et al. (2020) Rapid colorimetric detection of COVID-19 coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic plat-form: iLACO. medRxiv, 10.1101/2020.02.20.20025874.

2. Butt,A.M., Siddique,S., An,X. and Tong,Y. (2020) Development of a dual-gene loop-mediated isothermal amplification (LAMP) detection assay for SARS-CoV-2: A preliminary study. medRxiv, 10.1101/2020.04.08.20056986.

3. Lu,R., Wu,X., Wan,Z., Li,Y., Zuo,L., Qin,J., Jin,X. and Zhang,C. (2020) Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2. Virol. Sin., 10.1007/s12250-020-00218-1.

4. Carter,C., Akrami,K., Hall,D., Smith,D. and Aronoff-Spencer,E. (2017) Lyophilized visually readable loop-mediated isothermal reverse transcriptase nucleic acid amplification test for detection Ebola Zaire RNA. J. Virol. Methods, 244, 32–38.

5. Wang,Z., Gu,Q., Sun,H., Li,H., Sun,B., Liang,X., Yuan,Y., Liu,R. and Shi,Y. (2013) One-step reverse transcription loop mediated isothermal amplification assay for sensitive and rapid detection of Cucurbit chlorotic yellows virus. J. Virol. Methods, 195.

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