Introduction

Infectious disease outbreaks of rapid spreading are common among populations of farm animals in China and all over the world. Hence, researchers have since long striven to develop simple and fast methods for virus detection that could be deployed on field without the need for advanced laboratory equipment. An illustrative example is presented by JinLong et al. 2010 for the case of Goose Parvovirus in goslings. The present COVID-19 outbreak poses a similar change, but in this case for the human population across the globe. In these cases, early detection of infected elements and their separation from the healthy population is crucial to contain the spreading of the virus. However, the magnitude of the COVID-19 pandemic makes it virtually impossible to provide access to testing for every citizen. Although, the production of COVID-19 test kits in undergoing a scaling up process, the qualified human resources needed to conduct the tests is much more difficult to scale up. Therefore, in the current conditions, a number of pre-screening criteria have been put in place before allowing access to COVID-19 tests. Among the main criteria, we can highlight the need to show symptoms. However, the likelihood of pre-symptomatic or asymptomatic individuals spreading the virus remains high. No doubt then that enabling massive daily self-testing of the population will have a tremendous impact on limiting the spread of the virus. Self-testing is an ability unique to the human population. It is not found in other animal populations. Our ability to exploit this advantage in the fight against COVID-19 could make the difference, and enable a new approach to the pandemic until vaccine development.

The problem our project solves

Daily at home or at work testing for covid-19 is a dreamt scenario that would have a tremendous impact in our ability to tackle the pandemic. This kind of intensive testing is not needed in most of existing type of viral infection diseases like HIV, flu, ebola, etc. where the comparatively small number of cases can be handle with laboratory tests. Moreover, the complexity of PCR test technology and the shortage in testing reagents makes it impossible to undertake such task.

The solution we bring to the table

In the path toward developing simple and fast methods for virus detection, researchers have found a reliable companion on the LAMP technique. Loop-mediated isothermal amplification (LAMP) of viral DNA has enabled researchers to detect viruses on the field making use of little lab equipment and in a fast way. The LAMP technique is not so versatile as PCR (Polymerase chain reaction), but it serves the purpose of simple on-field virus detection. This is mainly due to its isothermal characteristic. While PCR requires advanced equipment for its thermal cycle, LAMP requires just a simple water bath or heating block with temperature around 65 degree Celsius. Very recently, Zhang et al. (2020) have reported a protocol for the detection of SARS-CoV-2 (COVID-19) virus using LAMP and have highlighted the real possibility of developing portable devices for the detection of Covid-19 using this protocol. Moreover, quite recently, To et al. (2020) reported the consistent detection of COVID-19 in human saliva. Although the mechanism by which COVID-19 reaches human saliva is still unknown, this fact makes saliva a great candidate for a sample fluid in COVID-19 daily self-testing. A COVID-19 daily self-testing device or kit would allow extraction of the DNA material from a saliva sample, amplification of the viral DNA using the COVID-19 primers, dyeing of the LAMP product with EvaGreen®, and identification of the positive reaction by colour change that is visible to the naked eye (change from yellow to green). This type of devices are redundant for dealing with many types of viral infections, but in the case of highly contagious, fast-spreading, and life-threatening viral infections like COVID-19 they become essential.

The technology

RNA/DNA extraction: To et al. (2020) reported the consistent detection of COVID-19 in human saliva. A median viral load of 3.3·106 copies per ml was found in the first available saliva sample upon hospitalization. In some patients, a viral load of 1.2·108 copies per ml could be found. In order to proceed with the LAMP reaction, the RNA/DNA material (viral, bacterial, human, etc.) needs to be separated from potential LAMP reaction inhibitors also present in the saliva sample. Garbieri et al. 2017 studied DNA extraction from saliva using several methods or protocols. This methods, however, require centrifuges or vacuum manifolds. A more suitable method should be using silica coated magnetic beads for concentrating RNA/DNA from human saliva (Bordelo et al. 2013). This is done by selective capture of RNA/DNA using reversible binding to SiO2 coated paramagnetic beads.

RT-LAMP reaction for COVID-19: In the upstream process of the LAMP reaction we have the RNA to DNA conversion by employing reverse transcriptase. Thus, the RT-LAMP reaction can proceed if a number of primers are available, namely, the forward outer primer (F3), the backward outer primer (B3), the forward inner primer (FIP), the backward inner primer (BIP), the forward loop primer (LF), and the backward loop primer (LB). The RT-LAMP assay can be performed using a water bath or heating block at constant temperature (60–65 °C) for isothermal heating (Silva et al. 2019). Loop-mediated isothermal amplification (LAMP) was developed by Notomi et al. (2000) as a simple and rapid gene amplification technique. The method uses multiple primers designed to create continuous loop structures during DNA amplification. With the inclusion of reverse transcription (RT), the RT-LAMP method can be applied in detection of RNA viruses like Covid-19. If a device for Covid-19 self-testing at home/work can be conceived, it will certainly be with LAMP.

LAMP products detection with EvaGreen®: The amplified products can be detected by using the EvaGreen®. This dye is not fluorescent by itself, but becomes very fluorescent upon binding to dsDNA. The dye is quite stable both hydrolytically and thermally. If LAMP products of the targeted virus are present in the assay container, the EvaGreen® dyed sample turns from yellow to green, if not it remains yellow. This change can be detected by naked eye.

The Prototype

Since we want to deliver a device for Covid-19 self-testing at home/work, a primary functionality of such device is limiting contact between the user and the chemicals employed in virus detection. This is not a major issue in the case of qualified users in lab settings, but is an important one for unqualified users in a home/work setting. Thus, a low cost and easy to manufacture integrated device is to be designed and 3D printed. This device will allow for all the technical steps of the virus detection process, will limit the contact between the chemicals and the user, and will allow to observe the change in color upon completion of the test. We will also explore the possibility of enabling pool testing with our device. We will pursued the validation mechanisms put in places by EU authorities for Covid-19 diagnosis tests and we will enable suitable mechanisms for safe disposal of the used tests so to avoid any negative environmental effects.

What we have done during the weekend

We had a very productive weekend. During Saturday April 25, we discussed our project with our EuvsVirus Hackathon mentor Aaron Cabrera. Together we came up with ideas to improve the project and our video pitch. We also got many suggestions from other mentors and participants taking part in the hackathon. On Sunday, we got expressions of interest from several European companies like ArrowFast and Angel Consulting SAS for providing support to our project, which we welcomed. All the checkpoints helped us to reach the final submission in great shape and with many improvements.

Our solution’s impact to the crisis

Being able to separate infected individuals from the non-infected population is part of the ‘ABC’ for fighting viral epidemics affecting non-human or human populations. However, limited resources usually makes massive daily testing an impractical approach to contain pandemics. Hence, techniques like LAMP that require less complex laboratory equipment are great candidates for developing testing solutions enabling massive daily self-testing in populations affected by diseases like Covid-19. A portable device for Covid-19 self-testing at home/work could enable many millions of people regularly testing themselves and their close ones. We aim providing 10 million tests per month during our first 2 months of operation, with each test kit worth 1 month of daily testing per person. By our 3rd month of operation, we aim to reach a production level of 100 million tests per month. While by our 4th month of operation, we aim bringing our production to a level of 1100 million tests per month. This huge level of test kits production only can be reached with simplified technologies like LAMP. Coupling this technology with efficient manufacturing methods like 3D printing, and an optimal integration with the elements down in the supply chain represent a solid base for the feasibility of this bold approach. Unique challenges like Covid-19 require unique and moon-shoot solutions like this we are presenting.

The necessities in order to continue the project

Capital requirements: We are aiming at a pre-seed investment of euro 35000 for prototype completion, and a seed investment of euro 450000 for device regulatory approval and initial commercialization. These investments would allow us testing the concept, developing the prototype device, and move ahead with regulatory approvals, official validation, and initial commercialization. We expect a revenue of 4.15 million euro upon initial commercialization of the device in a market that is requiring millions of covid-19 tests per day.

Human resources: We are aiming an initial team of seven members who will support the projects in aspects like investment, marketing, regulatory affairs, 3D printing, among others.

Timeline: We will require a period of 6 weeks for device development, 4 weeks for regulatory validation, and 3 weeks for initial commercialization.

The value of our solution after the crisis

This type of technology will have a strong value during and after this crisis. For many years, LAMP have been identified as a technique with a tremendous potential to enable fast massive daily testing of non-human populations affected by acute viral outbreaks in farms or nature, and COVID-19 has made it evident that its potential also extends to the case of human populations. An investment made now in this methodology for diagnostics of viral infections will pay off greatly now and in the future ahead. This will improve our capability to keep pandemics at bay and to avoid the disastrous economic impact they have when they affect either human as non-human populations.

References

Bordelon, H., Russ, P. K., Wright, D. W., & Haselton, F. R. (2013). A magnetic bead-based method for concentrating DNA from human urine for downstream detection. PloS one, 8(7), e68369. https://doi.org/10.1371/journal.pone.0068369

Garbieri, T. F., Brozoski, D. T., Dionísio, T. J., Santos, C. F., & Neves, L. T. (2017). Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols. Journal of applied oral science : revista FOB, 25(2), 147–158. https://doi.org/10.1590/1678-77572016-0046

Kelvin Kai-Wang To, Owen Tak-Yin Tsang, Cyril Chik-Yan Yip, Kwok-Hung Chan, Tak-Chiu Wu, Jacky Man-Chun Chan, Wai-Shing Leung, Thomas Shiu-Hong Chik, Chris Yau-Chung Choi, Darshana H Kandamby, David Christopher Lung, Anthony Raymond Tam, Rosana Wing-Shan Poon, Agnes Yim-Fong Fung, Ivan Fan-Ngai Hung, Vincent Chi-Chung Cheng, Jasper Fuk-Woo Chan, Kwok-Yung Yuen, Consistent Detection of 2019 Novel Coronavirus in Saliva, Clinical Infectious Diseases, , ciaa149, https://doi.org/10.1093/cid/ciaa149

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.

Silva, S.J.R.; Pardee, K.; Pena, L. Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review. Viruses 2020, 12, 19.

Yinhua Zhang, Nelson Odiwuor, Jin Xiong, Luo Sun, Raphael Ohuru Nyaruaba, Hongping Wei, Nathan A Tanner. 2020. Rapid Molecular Detection of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv. doi: https://doi.org/10.1101/2020.02.26.20028373

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