An automated nucleic acid detection platform using digital microfluidics with an optimized Cas12a system

Abstract

Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics (DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection. The RCD platform demonstrated a high level of sensitivity, specificity (no false positives or negatives), speed (≤30 min), automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of qPCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics. Electronic Supplementary Material Supplementary material is available in the online version of this article at 10.1007/s11426-021-1169-1.

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…In the single tube reaction, the amplification template loss caused by RPA-CRISPR/Cas12a cleavage would lead to low detection efficiency, Lin et al found that glycerol additive could significantly improve the detection efficiency of one-pot RPA-CRISPR/Cas12a, and its sensitivity was nearly 100 times higher than that of the method without glycerol. This optimized RPA-CRISPR/Cas12a had been successfully used to detect SARS-CoV-2 and ASFV (Lin et al, 2022). During the establishment of a one-pot detection method based on RPA-CRISPR/cas12a, the development and optimization of methodology could be accelerated by using the statistical design of experiments (Malci et al, 2022).…”

“…RCD platform showed high sensitivity and specificity and could realize automation and multiplexing. It has been successfully used to detect influenza virus and SARS-CoV-2 (Sun Z et al, 2022).…”

Microfluidic chip and isothermal amplification technologies for the detection of pathogenic nucleic acid

“…DMF has broad application prospects in biology due to its advantages such as portability, reagent saving, and reduced power consumption. Sun et al developed an automated nucleic acid detection platform (RCD) for influenza viruses and SARS-CoV-2, which was based on optimized RPA-Cas12a combined with DMF [ 63 ] (Fig. 12 ).…”

“…c Side view of the DMF chip including a droplet. d Schematic illustration of the RPA-Cas12a-crRNA recognizing target and cleaving the reporter [ 63 ] …”

Construction of an Ultrasensitive Molecularly Imprinted Virus Sensor Based on an “Explosive” Secondary Amplification Strategy for the Visual Detection of Viruses

“…The history of human development is also the history of its fight against viruses, and countless people died in this endless struggle. − The rapid and highly sensitive detection of viruses is a key factor in reducing losses and ultimately winning the war against viruses, and this effort has drawn widespread attention from governments and scientists worldwide. − Various strategies for the detection of viruses have been developed and achieved fruitful results. These include electrochemistry, , Raman spectroscopy, − quartz crystal microbalance (QCM), , the polymerase chain reaction (PCR), , the enzyme-linked immunosorbent assay, , resonance light scattering (RLS), fluorescence spectroscopy, , and so on.…”