The team of Bai Jingwei, Li Yinqing and Liu Peng of Tsinghua University has jointly developed an all-in-one self-service SARS-CoV-2 nucleic acid detection cartridge, which can be tested within 30 minutes.
In the past, nucleic acid testing often required 2 to 3 hours of tedious operation and reaction under harsh environmental conditions and specialized equipment to obtain the results.
In addition, the sampling process is very susceptible to impurities, which can lead to loss of nucleic acid extraction, and the experimental conditions can also lead to low amplification rate, which often results in "false negatives".
This time, the nucleic acid detection cassette developed by Bai Jingwei and Li Yinqing from Tsinghua University School of Pharmacy and Liu Peng from Department of Biomedical Engineering, Tsinghua University School of Medicine overcame the stringent requirements on time, conditions and sensitivity of nucleic acid detection in the past, and realized the self-help operation from pharyngeal swab sampling, integrated cassette reaction and colloidal gold reading.
"Time-boosting is the biggest problem we have to solve this time for nucleic acid testing!" Bai Jingwei has been engaged in gene sequencing technology for a long time and has accumulated rich experience in nucleic acid sample collection, processing and testing. He analyzed that nucleic acid detection PCR method uses the traditional way to let the sample amplify, at first the sample replicates quickly, but later the speed decreases and takes too long, "We used nest isothermal amplification technology in the technical research."
Bai Jingwei likened the process of amplification of the constant nucleic acid samples for the new coronavirus detection to an "athlete's long run", in which an athlete starts out fast, but often lacks endurance.
The same is true for sample amplification, where replication is fast at first, but decays halfway through, so to increase speed, they changed the "long run" to a "relay run", which can detect single-digit novel coronaviruses in a short time.
"Finally, the above reactants were colored on colloidal gold test paper by the competition method and read in a manner similar to a pregnancy test stick, at a glance." Bai Jingwei said the process can theoretically be completed in 22 minutes, while "sample in, result out" can be achieved within 30 minutes if the individual tester operates.
To overcome the "high false-negative" problem of traditional assays, Li Yinqing said they have integrated and streamlined sample acquisition, nucleic acid amplification, pathogen identification and colloidal gold immunochromatography to reduce the intervention of testers and improve the sensitivity of the assay.
"In terms of simplifying sample processing, for example, because pharyngeal swab samples often contain reaction-inhibiting impurities, routine assay samples often go through a nucleic acid extraction process that is time-consuming and relatively manual and not suitable for untrained personnel."
By optimizing the lysis process and back-end amplification reagents to allow direct access to reverse transcription isothermal amplification without nucleic acid extraction, the team greatly reduced the complexity of the detection process and reduced the number of "false negatives" due to impurities.
Liu Peng said that in order to facilitate detection, they have also created an "integrated microfluidic cassette", which is divided into four steps.
In the first step, a moving rod drives the syringe-type plunger causing the cleaved sample to flow into the first isothermal amplification chamber.
In the second step, the reaction product is diluted after 10 minutes of reaction.
In a third step, pump into a second isothermal chamber for further amplification with probes.
Finally, the reaction mixture is pumped into a chamber containing laminar flow test strips to read out the test results.
In this, the cassette contains a simple USB or battery-powered thermostatic circuit that provides the temperature required for the amplification reaction.
Liu Peng mentioned that in order to improve the user experience of the cassette, they invited Zhao Chao, vice president of Tsinghua Academy of Fine Arts, to design the structure and appearance of the cassette, "It is expected that the future cassette will be about 10x10x3cm in length and width and height, when the inspector uses the cassette, he only needs to push up and down on each of the two pushers, and the results can be viewed in 30 minutes, the operation is very simple."
It is envisioned that the team will also develop batch test kits, perform multiple sample tests, and develop a mobile APP to assist users in using and interpreting the results.
The project team will advance small batch cassette assembly to validate the feasibility of mass production, while working with hospitals to accelerate the validation of clinical samples. Currently, the team is also actively looking for future partners to expedite the approval of the inspection cartridge.
Special Report: Fighting The New Coronavirus
