Clustered regularly interspaced short palindromic repeats (CRISPR) has shown significant promise as an emerging nucleic acid detection technology. However, it still requires improvement in terms of sensitivity, detection automation, and anti-pollution. Furthermore, CRISPR technology lacks simple and portable professional equipment to meet the high demand of rapid point-of-care testing. Therefore, this study proposes a CRISPR/Cas12a detection reaction system for SARS-CoV-2. This detection response system and innovative tube-in-tube consumables aid in developing a portable compact device for simultaneous automatic detection of several samples and a coaxial fiber-based fluorescence detection system. Finally, we developed a single-sample user-friendly nucleic acid detection APP based on smartphone recognition and detection results for the manual detection mode.
The target in this study was severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which was detected using the CRISPR method and enhanced via the reverse transcription-recombinase polymerase amplification (RT-RPA) technique; the feasibility was assessed using the reverse transcription-polymerase chain reaction (RT-PCR) amplification method in the early stages. Various companies customized the required reagents and the designed sequences. In the detection process, first, with the tube-in-tube consumables developed by our team in the early stage, which comprised the reaction outer and inner tubes, the amplification reagents and detection reagents were loaded into the inner and outer tubes, respectively. The temperature was regulated to 37-42 ℃ to complete the amplification. The reagents in the inner and outer tubes were then mixed by shaking or centrifugation, and the temperature was adjusted to complete the CRISPR reaction. Finally, it was possible to observe if there was any fluorescence occurrence under the illumination of a blue light. The detection instrument was composed of an optical cassette and a base, and automatic detection was realized through a printed circuit board (PCB), a human-computer interaction display screen, etc. In addition, this study also used the fluorescence image recognition algorithm to process the detection images, compared with the international standard polymerase chain reaction (PCR) technology to explore the detection limit, and increased the target types to test the specificity strength.
The lower part of the detection instrument designed by our team integrates the printed circuit board and the human-computer interaction display screen. In the automatic detection mode, the fluorescence recognition circuit was designed with the help of a 470 nm light-emitting diode (LED), an optical filter, a complementary metal oxide semiconductor (CMOS) camera, a collimating lens, and a coaxial fiber. At the same time, the specificity of the theoretical experiment was verified through comparative experiments on several different targets. In addition, to verify the accuracy of this method for detecting actual samples, we compared each actual sample through PCR detection and the method based on the combination of RT-RPA and CRISPR proposed in this study. The detection results showed that the two were perfectly consistent.
The current study proposed a CRISPR/Cas12a-based anti-pollution portable nucleic acid detection technique. Furthermore, a simple model was proposed based on the naked eye or smartphone to recognize results; additionally, a downsized portable device based on fluorescence detection that can simultaneously detect numerous samples was constructed. The portable device can detect numerous samples simultaneously, and it has a constant heating mechanism and fluorescence stimulation detection optical channel to enhance the detection system’s accuracy and stability. The nucleic acid of SARS-CoV-2 was verified using the proposed method and detection system. The minimum detection limit was <10 copy/μL. The test findings of our method had a good consistency with that of real-time fluorescence quantitative PCR method, but our method took less than half the time consuming of the PCR method, and the whole detection process could be finished in 32 min. The method and technology developed in this study propose a novel approach for nucleic acid detection at health-care center and home.
