Combined recombinase polymerase amplification/rkDNA-graphene oxide probing system for detection of SARS-CoV-2

Abstract

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.

Keywords: Enzymatic in situ synthetic probe (rkDNA); Fluorescence; Graphene oxide; Isothermal amplification; RPA; SARS-CoV-2.

Graphical abstract

Scheme 1

Combining RPA with an rkDNA–GO system for the detection of COVID-19.

Fig. 1

(a) Denaturing PAGE (polyacrylamide gel electrophoresis in Urea) of the RPA for 1 nM of N-SARS-CoV-2 and N-SARS-CoV with RPA-Rev Primer and RPA-Fow Primer: lane 1: 1 nM of N-SARS-CoV-2 + RPA-Rev Primer + RPA-Fow Primer; lane 2: 1 nM of N-SARS-CoV-2 + RPA-Rev Primer + RPA-Fow Primer + RPA enzyme kit; lane 3: 1 nM of N-SARS-CoV + RPA-Rev Primer + RPA-Fow Primer; lane 4: N-SARS-CoV + RPA-Rev Primer + RPA-Fow Primer + RPA enzyme kit. (b) Non-denaturing PAGE confirming the cleavage of the extended reverse primer by Lambda exonuclease obtained through RPA lane 1: product of RPA reaction with N-SARS-CoV-2; lane 2: product of RPA reaction with N-SARS-CoV2, treated with Lambda exonuclease. The gels were stained using SYBr gold. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Sensitivity of combined RPA/rkDNA–GO probing system for the detection of N-SARS-CoV-2. (a) Fluorescence response (λ_em = 545 nm) with error bar of the RPA/rkDNA–GO probing system in the presence of N-SARS-CoV-2 (1 fM–1 nM). (b) Linear relationship between the fluorescence response and the logarithm of the concentration of the target N-SARS-CoV-2 (from 1 fM to 1 nM). F is fluorescence response of different concentration target and Fo is fluorescence intensity of rkDNA-GO. Each sample contained 0.1 μM (1 mL) of rkDNA in 25 mM Trizma buffer (25 mM Tris, 50 mM NaCl, 10 mM MgCl₂) and 0.18 mg of GO at pH 7.2. Excitation wavelength: 410 nm.

Fig. 3

Selectivity of the combined RPA/rkDNA–GO probing system for 1 nM N-SARS-CoV-2 over 1 nM N-SARS-CoV, determined by their fluorescence responses at λ_em = 545 nm. Each sample contained 0.1 μM (1 mL) of the rkDNA in 25 mM Trizma buffer (25 mM Tris, 50 mM NaCl, 10 mM MgCl₂) and 0.18 mg of GO at pH 7.2. Excitation wavelength: 410 nm.

Fig. 4

Sensitivity of the combined RPA/rkDNA–GO probing system for the detection of N-SARS-CoV-2 when performing the RPA reaction for 1 h. (a) Concept for RPA amplifying by time. (b) Selectivity of the RPA/rkDNA–GO probing system for 1 fM N-SARS-CoV-2 and 1 fM N-SARS-CoV when using an RPA reaction time of 1 h, determined by the fluorescence response at λ_em = 545 nm. (c) Fluorescence response (λ_em = 545 nm) with error bar of the RPA/rkDNA–GO probing system in the presence of N-SARS-CoV-2 (1 aM–10 fM). (d) Linear relationship between the fluorescence response and the logarithm of the concentration of the target N-SARS-CoV-2 (from 1 aM to 10 fM). F is fluorescence response of different concentration target and Fo is fluorescence intensity of rkDNA-GO. Each sample contained 0.1 μM (1 mL) of rkDNA in 25 mM Trizma buffer (25 mM Tris, 50 mM NaCl, 10 mM MgCl₂) and 0.18 mg of GO at pH 7.2. Excitation wavelength: 410 nm.

Fig. 5

Results of spiked specimens using RPA/rkDNA-GO probing system. Blank is rkDNA-GO without target. Significantly increased fluorescence intensity in positive samples were observed.