This year, we developed a device combining synthetic biology and other disciplines, and implemented it into a virus detection project, to establish the G-quadruplex directed colorimetric virus detection system. We want to build a platform for real-time visual detection of various viruses and their variants, which may also be applied to the detection of pathogenic bacteria in future. Our design can achieve high specificity and sensitivity, and the involved reactions can be basically completed under isothermal conditions, which greatly reduces the need of the resources for the detection in the fields or non-laboratory circumstances. As an isothermal DNA amplification technique, the rolling cycle amplification (RCA) has proved to be a versatile tool. We want to detect viruses and their variants with high accuracy and sensitivity. We chose saliva as a convenient sampling approach. After the samples are collected, the protein shell of the virus is first removed by protease digestion; then, the RNAs for specific detection were extracted by a simple spinning and heating device, and finally the dsDNA was obtained by an isothermal reverse transcriptase, followed by the G-quadruplex detection system.
Our device is manually controlled for the reaction of each step, divided into one, two and three gears. The reaction is sequentially controlled to ensure the accuracy of the test results. Compared to the existing virus detection system, our device holds multiple advantages. The heating module controls the temperature, which allows the test to be carried out in outdoor conditions. The device has a real-time detection and visualization with a clear and discernible color change from transparent to green, if the virus is detected. Therefore, our device can be widely used, especially in the scenarios of desolate areas without access to standardized laboratories. Our device is tightly sealed to prevent the test samples from leaking into the external environment. Nevertheless, we still realize its potential safety risks, including possible virus leakage during the sample extraction process. Due to the short sampling time, the possibility of this leakage is very minor. Our device still faces certain challenges, such as the cryogenic storage of enzymes during transportation, and storage conditions of samples and the whole device. Regarding the enzyme storage, we can use their lyophilized powder in transportation, but the samples storage still remains a challenge to us.
Currently, chemiluminescence is the major approach used for antibody detection in the market, but its platform is highly demanding for advanced equipment and a clean operating environment. In the applications of nucleic acid detection, the major advantage of chemiluminescence is its easy sampling and operation, while its disadvantage is the relatively slow detection. For general screening or the testing in non-developed countries, detection methods based on the colloidal gold have relatively low requirement for personnel, sites and equipment, and are therefore suitable for rapid and large-scale detection. However, many countries do not have technical reserves in colloidal gold production or usage. Actually, most global supplies of colloidal gold-based tests for COVID-19, influenza, malaria and venereal diseases are still in China. Therefore, our device will not only circumvent the strict demand of chemiluminescence tests for the equipment and operating environments, but also improve the situation of relatively slow detection.
Currently, the traditional method of viral nucleic acid detection generally requires more than 2 hours, which is basically the time used in nucleic acid amplification. Nucleic acid detection can only be achieved after the detected nucleic acid sequence in a sample reaches a certain number through its amplification, which typically includes three steps of denaturation, annealing and extension. In the quantitative fluorescence PCR used in traditional nucleic acid detection, these three steps need to be carried out successively at different temperatures. The temperatures for the denaturation, annealing and extension steps are typically 95℃, 60℃ and 72℃, respectively. If all three steps can be merged into one, the testing time will be greatly reduced. In our device, we employed the isothermal DNA amplification technology that can significantly decrease the reaction time. With amplification being carried out under ambient temperatures, it is equivalent to the combination of the three steps of the PCR amplification. With further optimization, the reaction of RCA-based nucleic acid amplification can be completed within 30 minutes.
As a type of microorganisms, infectious viruses threaten the global economy, human lives and health in many ways. Prevention of pathogenic virus infection is always the top priority in our society, and virus detection is a very important part. For the prevention and control of infectious viruses, the development of detection methods with cheap and easy indicators is always an important and arduous task. With continuous improvement of our testing method, we hope that our device can greatly advance the virus detection technology in future.
