Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores

Abstract

The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.

Detection of small-sized DNA fragments in a glassy nanopore by utilization of CRISPR-Cas12a as a converter system

“…The integration of the CRISPR-Cas12a and nucleic acid amplification techniques has been proved by several works. 22,60,63–65 These results demonstrated that our nanopore sensor using a DNA tetrahedron as a signal transducer based on the CRISPR-Cas12a conversion mechanism could detect targets specifically, which we believe could become a promising alternative to biomolecular detection.…”

“…52,55–58 However, the application of CRISPR-Cas12a in nanopore sensing has been reported only recently. 56,59,60…”

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

“…Sensitive readout systems with lower C min could help achieve lower FOM and better sensing performance (eq 5). While the majority of Cas12 or Cas13-based sensing systems were based on fluorescence signal, 11,16,77 colorimetric, 15,78 electrochemical, 14,21 and electronic readout 45,79 were also explored for signal readout. Figure 2e compares the reported C min of different readout systems.…”

Use of MALDI-TOF mass spectrometry for virus identification: a review

“…8 Kudr et al 4 demonstrated the potential use of electrochemical biosensors for virus identification in their review. In many studies, loop-mediated isothermal amplification (LAMP) [9][10][11][12] and highly multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection [13][14][15][16] have been investigated as potential tools for SARS-CoV-2 detection. In addition to the above-mentioned diagnostic approaches, matrix-assisted laser desorption and ionisation with time-of-flight analyser mass spectrometry (MALDI-TOF MS) for SARS-CoV-2 detection was also demonstrated as another promising alternative in recent studies.…”