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

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.

Scheme 1

Reaction mechanism of SARS-CoV-2 RNA detection based upon Cas12a-assisted RTF-EXPAR reaction.

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

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

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

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

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.