When we first thought of developing a viral detection test, we wanted our project to be as close to commercially viable as possible. In order to do this, throughout the course of our project, we constantly conducted and referred back to our human practice research to guide the implementation of our project. This research influenced our project all the way from our construct design to our marketing plan.
Team:TAS Taipei/Implementation
Implementation
Using RCA (Rolling Circle Amplification) as the Detection Methodology
To develop a test kit that is useful, we first conducted research on current detection methodologies. There are three main detection methods: antibody, antigen, and molecular. Through human practice interviews, we found that the accuracy of viral tests depends primarily through two components, sensitivity and specificity. These two components are closely intertwined with safety risks such as the possibility of false positives and false negatives. Thus, we specifically looked into the specificity and sensitivity of each testing method and the safety involved with them.
Antibody and antigen rapid lateral flow tests have severe limitations in sensitivity and a resulting high rate of false negatives (Gordon & Michel, 2008; Rapid Influenza Diagnostic Tests | CDC, 2019). These inaccurate diagnoses may pose harmful impacts to not only the individual but also the community. For example, one who experiences a false positive diagnosis might undergo treatment for a virus they actually don't have, posing possible harm to the person. One who experiences a false negative diagnosis may be free to wander back into the human community, where the individual could infect others.
Mitigating the downsides of such test-kits, we use the Rolling Circle Amplification (RCA) as a new detection method. RCA has the positive attributes of the highly sensitive and specific RT-PCR tests, which continues to serve as the gold standard of viral testing (da Costa Lima et al., 2013; Giri et al., 2020; Tahamtan & Ardebili, 2020). By employing a similar nucleic acid testing technique, we further improved specificity through direct RNA targeting, dodging the use of possible low fidelity reverse transcriptase enzymes (Alhassan et al., 2015; Roberts et al., 1988; Skalka & Goff, 1993). However, we also took on the emerging field of isothermal amplification techniques in nucleic acid testing. But we went as far as to enable the reaction to be conducted at room temperature, not merely at extreme constant temperatures that might require thermocyclers, incubators, or other instruments.
Sensitivity and Specificity
As sensitivity and specificity are important for measuring the usefulness of the test kits, we tested the sensitivity and specificity of our RCA tests following the FDA EUA guidelines, particularly the limit of detection and cross-reactivity sections (Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (Revised) - Immediately in Effect Guidance for Clinical Laboratories, Commercial Manufacturers, and Food and Drug Administration Staff, n.d.). Conducting serial dilutions from 0.0625uM to 0.027pM, our sensitivity results have shown that we can detect up to 0.027fM of RNA. Our specificity tests on the other hand have shown that we can distinguish between different viral strains due to different time of color change in the reaction.
Even the inclusivity and clinical evaluation sections in the guidelines have assisted our selection of target regions and padlock probe designs, the process of which are outlined in the experimental section (Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (Revised) - Immediately in Effect Guidance for Clinical Laboratories, Commercial Manufacturers, and Food and Drug Administration Staff, n.d.). These have helped us ensure that no matter a virus type’s year, location, and similarity to other strains, we can recognize the viruses we want to target and follow the FDA expectation that all viral sequences published can be detectable with our probes.
Using Saliva Collection Method
From our public survey, 75% of our surveyed population express that, given the choice, they would prefer using home test kits over testing at a medical facility, assuming that the tests performed the same. Thus, our project aims to develop a home test kit.
To design a home test kit, we first researched and decided how to collect viral sample. Our HP research has shown that nasopharyngeal swabs require specialized medical personnel with safety equipment and still tend to lead to higher risk of viral exposure to healthcare workers (Iwasaki et al., 2020; Nasopharyngeal Swab vs. Saliva for COVID-19 Diagnosis, 2020). Saliva collection on the other hand maintains relatively the same sensitivity as nasopharyngeal swabs for detection, all the while reducing the risk of transmission due to the ability for adolescents and adults to self-collect saliva at home (Jamal et al., n.d.). Understanding the severe discomfort and even danger of nasopharyngeal swab testing, we decided to employ a saliva collection technique.
For tests such as RT-PCR, nucleic acid isolation is also necessary. These are usually met with RNA Extraction kits that require membrane columns, buffers, and centrifugation (Tan & Yiap, 2009). Not only is this process time consuming but once again introduces highly technical experimentation and the possibility of error.
Home Test Kit
To transform our diagnostic methodology to a home-based test kit operable by anyone at home, our modeling team designed a syringe device for saliva collection and purification, along with a separate test tube to conduct the actual detection process. To allow the consumer to easily administer their own home test kit, the saliva collection syringe will have all testing reagents needed for RNA purification prefilled for immediate use. The PCR tube will also include all the reagents needed to run the RCA.
To meet the safety, accuracy, comfort and usability in the sample collection and purification process, our modeling team created a two in one hand-held device that can achieve these goals. Focusing on allowing the sample to be prepped fast and without any background expertise, we envision this device to be used in every household. The design and theory behind the hardware piece can be found in the modeling hardware section.
Spitting saliva into the mouthpiece on the syringe device, pushing and pulling on the plunger, and eluting the isolated nucleic acid into the detection test tube, our color change will indicate the presence or lack of a virus.
As the used test kit could be unsafe due to sample collection of possibly infected individuals, the manual will come with instructions regarding how to correctly throw away the device after use. There are three steps to which customers should follow with the disposal of the used device. The customers should first put the device in a disinfecting solution for three hours, wash the device thoroughly with soap and water, and lastly microwave the device to ensure safety (Disinfection Technology and Strategies for COVID-19 Hospital and Bio-Medical Waste Management, n.d.) Since no elements in the test kit is biologically hazardous, the package label does not need to follow CDC’s guideline of biologically hazardous objects (CDC LC Quick Learn: Recognize the Four Biosafety Levels , n.d.).
Figure 1: CAD of our syringe handheld device for simple viral sample collection and purification. For more details, please visit our hardware page.
Confirming Test Results at Home
Our RCA testing, coupled with our specially designed syringe device, generates qualitative results. In order to provide the patients with more information regarding their infection, we designed a software, VisualpH, to quantify the color-readout of RCA reaction.
VisualpH works by taking the color of the RCA reaction, compiling the color to hue data, and then converting the hue data into pH data. By analyzing the rate of pH change, the software can tell the viral load of the patient.
This software expands a lot of possibilities for virus testing, as it could be downloaded to a smart device and used in conjunction with the Viral Spiral testing kit to provide analyses on testing results. The software uses Python and seeing as Python provides a lot of cross compatibility, the software could be adapted to be put in a multitude of different devices, so that testing could be done entirely at home. The user would be able to examine their test results just by taking a photo of it, and an app will immediately return the details of the test, like pH and viral load.
Furthermore, given biological details about the user, such as symptoms or age, the app could also potentially predict future events and how severe the disease will be for that specific user. The benefits of this prediction, could be very beneficial to the prevention and response to pandemics, as patients who will in the end only experience benign effects do not need to overload the medical response system. On the other hand, the patients who are most seriously affected could be given more attention earlier, hopefully making a recovery for them faster and less expensive. When a disease progresses, it gets harder to treat, but giving early intervention to those who may end up very ill could save a lot of medical resources and expenses.
Marketing
Although our project is still at a prototype stage, we want to see how our test kit will perform on the current test kit market. Therefore, we developed a marketing plan (read our marketing plan here)
In our marketing plan, to see how our test kit compared to other test kits, we looked into antigen, antibody, and molecular tests and did a SWOT analysis on each of them to analyze their respective strength and weakness. Considering that our test kit has high specificity, is instrument-free, and fast, we think that the performance of the test kit will be competitive against the other test kits.
We also analyzed the market to define the target market for our test kit. With the U.S. Food and Drug Administration now having authorized eight home test kits as of October 2020, which represents a growing market for home test kits, the United States stands as an ideal market for our home-based test kit. Specifically, we believe that targeting the market in California could be beneficial, due to California having one of the highest climbing infection rates and governmental activities conducted to expand testing capability in both symptomatic and asymptomatic people. Home test kits allow the individual to test whether he/she has a viral infection without having to do doctors’ visits, which could potentially result in an infection. This not only enables immediate results for patients to obtain early treatment and lower the chance of developing later complications but also helps relieve the strain on hospitals caused by the surge in COVID-19 cases.
From our public survey, we learned that pricing is a crucial element in deciding the usefulness of a test kit. On our survey trip, multiple people asked the question of whether the health care system will cover the cost for our test kit before they could answer the survey questions. From this, we learned that price is an important factor in an individual’s decision to get tested or not, which will be even more of the case in the U.S. where medical services cost a lot of money. Therefore, in the marketing plan, we specifically discussed the pricing for our test kit and all the factors involved. Due to the wide range of test kit prices, we initially found it difficult to price our test kit, especially since our project is still in the prototype phase. With the advice of an investor, Ms. Susan Lin, we price our test kit at $30. This price is a lot cheaper than most PCR test kits and only slightly more expensive than the cheaper-end antibody/antigen test, making our test kit competitive as well as affordable in the current test kit market.
The question about health care also prompted us to think about how we should distribute our test kits. To make the test kit as available to our target market as possible, we plan to collaborate with the California government considering its active effort in expanding test kit capability within the state. Judging from the fact that the California state government has formed a new partnership with PerkinElmer, a test kit company, in August, we believe that the state government would be able to subsidize our test kit, allowing us to price our test kit at the price of $30 or lower and making the test kit more affordable.
Implementing Viral Spiral in the Real World
With our RCA design, hardware, software, and our marketing plan, we believe that our project could be safely and effectively implemented in the real world.
Figure 16: Flowchart of our Viral Spiral Project for Viral Diagnosis
References
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