Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2

Abstract

Continuous identification of suspected infectious cases is crucial to control the recent pandemic caused by the novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Real-time polymerase chain reaction (real-time PCR) technology cannot be implemented easily and in large scale in some communities due to lack of resources and infrastructures. Here, we report a simple colorimetric strategy derived from linker-based single-component assembly of gold nanoparticle-core spherical nucleic acids (AuNP-core SNAs) for visual detection of PCR products of SARS-CoV-2 ribonucleic acid (RNA) template. A palindromic linker is designed based on SARS-CoV-2 specific E gene to program the identical colloidal SNAs into large assemblies along with a distinct red-to-purple color change. The linker acts as a probe of SARS-CoV-2 RNA in conventional PCR reaction. In the presence of the correct template the palindromic linker, which is complementary to a short region within the target amplicon, is cleaved by 5'-exonuclease activity of deoxyribonucleic acid (DNA) polymerase. Cleavage of the palindromic linker during the amplification process inhibits the single-component assembly formation of SNAs. So, positive and negative viral samples produce simply red and purple colors in the post PCR colorimetric test, respectively. Evaluation of the samples obtained from cases with laboratory-confirmed SARS-CoV-2 infection revealed that our assay can rival with real-time PCR method in sensitivity.

Keywords: 5’-exonuclease activity; Colorimetric detection; Coronavirus disease 2019 (COVID-19); DNA-programmable assembly; Post-PCR detection; Spherical nucleic acids (SNAs).

Graphical abstract

Fig. 1

Simulative and experimental characterization of the designed single-component assembly system. (A) The polymer graph of the assembly-guided structure simulated by NUPACK Analysis Algorithms (left) and DNA sequences forming single-component assembly (right). (B) TEM image of flower-like structure of single-component assembly (obtained from13-nm AuNP core SNAs and the used palindromic linker). (C) A 3D schematic illustration of flower-like structure of single-component assembly. (D) Time-dependent changes in the UV–vis spectra of 13-nm AuNP- core SNAs in the presence of palindromic linker over 40 min at 25 °C (60 μL of 10 mM HEPES buffer, pH = 7.6, containing 0.45 pmol SNAs, 5 pmol linker and 400 mM of NaCl). The inset is a photograph of the dispersed colloidal SNAs as control and the aggregated SNAs after 10 min of the addition of palindromic linker. Dispersed SNA particles are red, whereas aggregated SNA particles are purple. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2

A) Scheme of the 5′-exonuclease activity of the DNA polymerase in conventional PCR which cleaves the linker DNA associated with programmable assembly of SNAs. During the annealing step, linker-DNA probe and amplification primers are simultaneously hybridized to the target template and during the extension step, linker-DNA probe is displaced and cleaved by DNA polymerase. (B) Schematic diagram of the PCR-based colorimetric method workflow for SARS-CoV-2 RNA detection.

Fig. 3

Laboratory-based optimization. (A) Setting up the annealing temperature of the PCR reaction. PCR products for different annealing temperatures (written at the top of each lane) were checked by 5 % agarose gel electrophoresis. (B) Effect of NaCl concentration on the colloidal stability of SNAs. Extinction ratio Ex₇₀₀/ Ex₅₂₀ was monitored as a function of NaCl concentration in the presence of 0.625 mM Mg²⁺ (Note that the UV–Vis spectra were recorded 10 min after adding NaCl and control was pure SNAs. Error bars are standard deviations n = 3). (C) Evaluation of assembly growth via time dependency of Ex₇₀₀/ Ex₅₂₀ ratio at the various amounts of linker. The Ex₇₀₀/ Ex₅₂₀ ratios higher than 0.8 are coincident with the red-to-purple color change which could easily be observed by the naked eye.

Fig. 4

Experimental verification of the basic idea behind the strategy. (A) The gel electrophoresis image of PCR products obtained for positive and negative control samples in the presence of 0.25 μM of the linker probe after 45 cycles of PCR. (B) Photograph of the color changes corresponding to the same PCR product samples after adding SNAs (picture was taken using a digital camera Nikon D7200). In gel electrophoresis experiment, 1 μM of the linker probe was implemented alongside the positive and negative control samples. Note, the positive control had been confirmed by real-time RT-PCR with the Ct value of 29.58, and the negative control was contained nuclease-free water instead of the target template.

Fig. 5

Real-time and post PCR analysis of amplification products corresponding to the five-step 10-fold serial dilutions of the positive sample 1. (A) Real-time amplification curves (the dilution factors are shown at the top of each curve). (B) The gel electrophoresis images and, (C) Photographs showing colorimetric response (pure linker probe was implemented alongside the serial dilutions).