The reason for us to create a G-quadruplex-directed colorimetric virus detection system is to develop a sensitive, economic and convenient device that can be used in relatively undeveloped areas for infectious virus detection. We sincerely hope that this device can greatly contribute to the global epidemic prevention and control. To achieve this goal, the device has to be simple and convenient, so that everyone could use it easily when following the instruction. Additionally, we must take into account the environmental conditions and operator's safety. We have reduced the cost of the reagents and the complexity of the system by selecting appropriate enzyme reactions and suitable hardware materials. We hope to highlight the advantages of our design through promoting the hardware.
Our hardware is mainly divided into five parts as shown in the figure, the seal cover, reaction tube A, reaction tube B, protective casing and base. This small device can complete the entire detection process: sample processing, isothermal amplification, colorimetric reaction, and visualizing the results. The reaction tube A is used to process a collected sample, and the content is then transferred to the reaction tube B, which is a special sampler. It has three units to store enzymes and reagents required for RPA, single-stranded DNA (ssDNA) displacement and RCA, as well as color reaction. A user only needs to simply add them in a designated order to complete the detection.
For the treatment of collected samples, we extensively explored literature to find appropriate procedures. A relatively mature scheme of saliva-based diagnosis for SARS-CoV-2 has been reported.  In this procedure, saliva samples were incubated with Protease K to disrupt the viral capsid, and then denatured the Protease K through heat inactivation. From this point, the reaction mix is subject to the reaction of reverse transcription-coupled quantitative PCR (RT-qPCR) with the sensitivity of detecting 6-12 viruses per microliter. The recombinase polymerase amplification (RPA) employed in our scheme has been verified in our experiments to achieve the sensitivity of detecting 1 copy of DNA template per microliter, which theoretically meets the requirements of sensitivity. Therefore, it may be a reasonable choice for us to take the advantage of collecting saliva samples.
For the follow-up reaction of the sample, we chose isothermal amplification. Although it can maintain reliability within a certain temperature range, our experimental results showed that the amplification efficiency could be significantly improved when the temperature was constant and optimal. Thus, we hope our device can be thermally insulated as well to achieve the best detection results.
Therefore, an important requirement for the device to meet is the precise control of its reaction temperature. The temperature of 95°C is required to inactivate the Protease K during sample processing, and a constant temperature of 37°C is also needed for the subsequent reactions. Since our device may be used outdoor or in some relatively undeveloped areas, we need to keep the operation as easy as possible and the cost as low as possible. We utilized a low-cost electric heating material: polyimide film (PI film) with customizable sizes and shapes. Through a temperature controller, it can sustain temperatures between 30 and 130°C. Due to its high-power density and fast heating, it is an excellent temperature control module very suitable to our project. The cost of PI film, temperature control module and power supply of each device is within 4 USD, falling in the acceptable range of our expectation.
The final step of the test is the color reaction, which is not very demanding for the hardware. The regular Eppendorf tubes used in laboratories can meet the requirements.
For the reactions involved in the whole testing process, we take their requirements into account in the hardware design to ensure that it will not attenuate any advantage of our detection device.
Disposal treatment after detection
For safety reasons, at the end of the detection, the user should follow the instruction to set the reaction mix in the device to the highest possible temperature allowing the inactivation any biocomponent. This step will ensure to prevent the potential dispersion of any biohazard material to the environment. We added this step to minimize the risk associated with the usage of our device.
We have received many suggestions and reminders during various activities of the human practice for this project, such as a very important safety issue: how to prevent the virus escape or leakage from the samples. To solve this problem, we have designed a mechanism for transferring samples without the need of opening the lid of the tube A.
Another suggestion is: we use nCas9 in our system, which needs sgRNA to guide its action. However, the presence of contaminant RNase may adversely affect the accuracy of our detection, and potentially cause false negative results. Based on this concern, we designed a sampler as a three-in-one module, without the need of repetitive lid opening and closing.
In our initial design, we did not take the reaction temperature into account, unless it reaches extremes, hoping to broaden the applicable extent of our device. However, an expert advised us that a relatively constant temperature range would reduce the variability and consequently increase the reliability of the detection process. Therefore, we chose to use electric heating to maintain the reaction temperature of the device.
- Vogels, Chantal BF, et al. "SalivaDirect: Simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance." MedRxiv (2020).