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. 2022 Nov 15:216:114683.
doi: 10.1016/j.bios.2022.114683. Epub 2022 Sep 6.

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

Xiao-Min Hang  1 , Hui-Yi Wang  1 , Peng-Fei Liu  2 , Kai-Ren Zhao  1 , Li Wang  3
Affiliations

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

Xiao-Min Hang et al. Biosens Bioelectron. .

Abstract

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/μL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/μL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.

Keywords: CRISPR-Cas12a; End-point fluorescence; Isothermy; RTF-EXPAR; SARS-CoV-2 RNA.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Scheme 1
Scheme 1
Reaction mechanism of SARS-CoV-2 RNA detection based upon Cas12a-assisted RTF-EXPAR reaction.
Fig. 1
Fig. 1
Viability analysis. (A) PAGE analysis of the RTF feasibility. (B) PAGE analysis of the EXPAR feasibility. (C) End-point fluorescence assay of the SARS-CoV-2 RNA detection strategy. The target product band was circled in the red box.
Fig. 2
Fig. 2
Optimization of experimental condition. Amplification time of EXPAR. The concentration of SARS-CoV-2 RNA: 100 fM. Error bar means SD (n = 3).
Fig. 3
Fig. 3
Analytical performance. (A) The overall operation of the strategy for SARS-CoV-2 RNA detection. (B) Evaluation of end-point fluorescence intensity in the presence of different concentrations of SARS-CoV-2 RNA (1 nM, 100 pM, 10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 100 aM, 10 aM, 1 aM and blank). (C) The linear relationship between Δ FL and the logarithm of the target concentration. Error bar means SD (n = 3).
Fig. 4
Fig. 4
Analytical performance (using “Palette Cam” app). The linear relationship between Δ Green intensity and the logarithm of the target concentration. Inset: Visual fluorescence intensity of SARS-CoV-2 RNA (different concentrations) involved in Cas12a-assisted RTF-EXPAR reaction under UV light irradiation. Error bar means SD (n = 3).
Fig. 5
Fig. 5
Specificity and practicability evaluation. (A) Evaluation of the specificity of the detection strategy. The concentrations of MERS-CoV and SARS-CoV: 10 pM. The concentration of SARS-CoV-2: 100 fM. (B) Δ Fluorescence, (C) Δ Green analyses of SARS-CoV-2 RNA transcribed in vitro standard material. Error bar means SD (n = 3). ns, nonsignificant.
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