CN114875180B - A DNA walker for SARS-CoV-2 detection and preparation method thereof - Google Patents

Abstract

The present invention relates to a DNA walker for SARS-CoV-2 detection and its preparation method. The DNA walker for SARS-CoV-2 detection comprises a walking chain W, a locking chain L1, a locking chain L2, a track chain Tr, a target chain T1, a target chain T2 and nano gold particles. The invention also provides a preparation method of the DNA walker and a method for detecting SARS-CoV-2 by using the DNA walker. The DNA walker provided by the invention realizes visual SARS-CoV-2 detection, and has the advantages of few interference factors, low price and good stability, and the sensitivity can reach 1nM. When the DNA walker or the kit is used for detection, a reverse transcription step is not needed, the use of enzymes, markers or modifications is avoided, the result is visualized, the requirement on complex equipment is reduced, and a convenient, economical, rapid and reliable method is provided for SARS-CoV-2 virus detection.

Description

A DNA walker for SARS-CoV-2 detection and preparation method thereof

Technical Field

The present invention relates to a DNA walker for detecting SARS-CoV-2 and a preparation method thereof, belonging to the technical field of biological detection.

Background technique

Since its first appearance, COVID-19 has spread rapidly around the world and become a global pandemic, posing a serious threat to public health. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus belonging to the Coronaviridae family. The disease is mainly transmitted through respiratory droplets and contact and is highly contagious. Rapid and reliable detection of SARS-CoV-2 is essential to prevent the spread of the epidemic and treat infected cases early. At present, the conventional detection method for SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR). However, this method requires specialized equipment to cyclically adjust the reaction temperature and monitor fluorescence in real time, and the operator needs to be professionally trained. And due to the limitations of qPCR detection equipment, the timeliness of detection and the huge detection volume are challenged, especially in routine or large-scale detection. At the same time, the turnaround time of RT-qPCR detection is slow, and the viral RNA should be reverse transcribed into complementary DNA before PCR. The reliability of RT-qPCR may also be affected by false positive results from nonspecific amplification of environmental pollutants or false negative results from the presence of amplification inhibitors.

In view of the shortcomings of RT-qPCR, isothermal amplification methods that do not require complex thermal cyclers have recently emerged for SARS-CoV-2 detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed using DNA polymerase and several primers for identifying different regions of the viral genome. In addition, the CRISPR-Cas system has also been adapted for the detection of SARS-CoV-2 sequences based on the nonspecific side chain ssDNAse activity of Cas proteins when target sequences are bound under the guidance of CRISPR RNA. Although amplification can be performed at a single temperature, the output signal is usually in the form of fluorescence, which means that at least an excitation light source is required. In order to make the signal easier to measure and even visualize the results, colorimetric methods have recently emerged for detection. RT-LAMP-based SARS-CoV-2 detection transduces the signal into a color change through a pH indicator that reacts to the acidic solution from the amplified DNA molecules, or by capturing protein-labeled DNA associated with RT-LAMP amplification to color the strips. In addition, for CRISPR-Cas-based SARS-CoV-2 detection, the output signal is transduced as a color change caused by the aggregation of gold nanoparticles. However, in these methods, isothermal amplification requires protein enzymes, which increases costs, requires strict storage conditions and has the risk of protein inactivation. And due to the unique activity of the enzyme on the DNA substrate, RNA still needs to be reverse transcribed. At the same time, because amplification is induced by single sequence binding, only one target nucleic acid fragment can be detected at a time, and the efficiency of confirming positive cases is low.

Recently, DNA molecular machines have shown promise in biomarker detection due to their biocompatibility, flexibility, and integrity. Among them, DNA walkers are DNA molecular machines that can perform step-by-step walks when stimulated. Such spontaneous walks are considered to be an inherent signal amplification method because a single recognition will lead to repeated walks, which helps signal accumulation. DNA walkers can move on one-dimensional linear tracks, two-dimensional planar tracks, and three-dimensional spherical tracks. For three-dimensional DNA walkers, it not only integrates the components into one nanoparticle or microparticle, but also gives the immobilized oligonucleotides the ability to resist nucleases due to the increase in local salt concentration around the particles. On the other hand, DNA logic gates can analyze multiple inputs and respond to outputs through Boolean logic operations, which provides convenience for processing different situations of the target. Various logic systems can be flexibly designed according to detection needs, such as the "AND" logic gate only outputs logical true values or obtains signals when both inputs are present.

Therefore, it is urgent to develop a convenient, economical and rapid SARS-CoV-2 detection method.

Summary of the invention

In view of the deficiencies in the prior art, the present invention provides a DNA walker for SARS-CoV-2 detection and a preparation method thereof.

A DNA walker for SARS-CoV-2 detection, comprising a walking chain W, a locking chain L1, a locking chain L2, a track chain Tr, a target chain T1, a target chain T2 and gold nanoparticles;

The nucleotide sequence of the walking strand W is: 5'-HS-(T) ₄₂ TGGTTCAATCTGTCAATCTCTTCTCCGAGCCGGTCGAAATAGTCCATAACCTTTC CACA-3';

The nucleotide sequence of the locked strand L1 is: 5'-TGCGGTATGTGGAAAGGTTATGGACTA-3';

The nucleotide sequence of the locked strand L2 is: 5'-AAGAGATTGACAGATTGAACCAGCTTGAG-3';

The nucleotide sequence of the track chain Tr is: 5'-HS-(T) ₆ GGCTGTGGACTAT/rA/GGAAGAGATTCAGCCCGCGTTTTTTTCGCG-6-Carboxyfluorescein (FAM)-3';

The nucleotide sequence of the target strand T1 is: 5'-CCATAACCTTTCCACATACCGCA-3';

The nucleotide sequence of the target chain T2 is: 5'-CTCAAGCTGGTTCAATCTGTCAA-3'.

Preferably, according to the present invention, the preparation method of the nano-gold particles is as follows:

A sodium citrate aqueous solution with a concentration of 38.8 mM and a HAuCl ₄ aqueous solution with a concentration of 1 mM were prepared; the HAuCl ₄ aqueous solution was heated to boiling, and then the sodium citrate aqueous solution was added to the HAuCl ₄ aqueous solution while stirring, and the mixture was reacted for 10 minutes under the heating boiling state. After the reaction was completed, the mixture was stirred for another 15 minutes, cooled to 25° C., and filtered to obtain gold nanoparticles (AuNPs).

A SARS-CoV-2 detection kit, comprising the above-mentioned DNA walker and _MgCl2 solution.

Preferably according to the present invention, the working concentration of the DNA walker in the kit is 3 nM, and the working concentration of the MgCl ₂ solution is 180 mM.

The preparation method of the above-mentioned DNA walker for SARS-CoV-2 detection comprises the following steps:

(1) The walking chain W, the locking chain L1 and the locking chain L2 are mixed evenly in an annealing buffer, then heated at 95°C for 10 min, and cooled to 20-30°C to obtain a completely closed walking chain; the completely closed walking chain is then incubated with TCEP for 2 h to obtain a thiol-completely closed walking chain;

(2) heating the track chain Tr at 95°C for 10 min to obtain an annealed track chain Tr;

(3) AuNPs, thiol-completely closed walking chains, and annealed track chains Tr were mixed evenly, and then incubated at 4°C in the dark for 16 h. Then, NaCl solution was added at intervals of 40 min until the final NaCl concentration was 0.2 M. The mixture was then incubated at 4°C in the dark for 24 h. After centrifugation and washing, a DNA walker for SARS-CoV-2 detection was obtained.

Preferably according to the present invention, in step (1), the molar ratio of the walking chain W, the locking chain L1 and the locking chain L2 is 1:3:3.

Preferably according to the present invention, in step (1), the molar ratio of the completely closed walking chain to TCEP is 1:50.

Preferably according to the present invention, in step (3), the molar ratio of the AuNPs, the thiol-fully closed walking chain and the annealed track chain Tr is 1:20:200.

The method for detecting SARS-CoV-2 using the above-mentioned DNA walker comprises the following steps:

The sample to be tested is added to a buffer solution with a final concentration of 3 nM of DNA Walker, reacted at 20-30°C for 2.5-3.5 hours, and then CaCl ₂ is added. When the incubation solution turns purple, the sample to be tested contains SARS-CoV-2.

Preferably according to the present invention, the final concentration of _CaCl2 in the buffer is 180mM.

The technical principle of the DNA walker of the present invention for detecting SARS-CoV-2 is as follows:

The inventors of this application selected two RNA gene fragments ORF1ab and N from the open reading frame and nucleocapsid of the SARS-CoV-2 viral genome as target chains T1 and T2. According to the target chains T1 and T2, the walking chain W, the locking chain L1, the locking chain L2 and the track chain Tr were designed, and then the DNA walker was synthesized in combination with AuNPs.

As shown in Figure 1, the DNA walker is a whole of DNA-modified AuNPs, the DNA is connected to the AuNPs through the end-labeled thiol group, and the AuNP surface is loaded with a small amount of walking chain W and a large amount of track chain Tr. The walking chain W contains a long swing arm region and a Mg ²⁺ -specific deoxyribozyme (8-17 type) catalytic domain. The locking chains L1 and L2 are not completely complementary to the target chains T1 and T2, respectively, so that the locking chains L1 and L2 each have a part of the single chain hanging outside, so as to identify and bind the target chains T1 and T2, and realize the replacement of the locking chain. The catalytic core of the walking chain W is closed on both sides by the locking chains L1 and L2. The absence of any one of the target chains T1 and T2 will cause the walking chain W to be unable to bind to the track chain Tr. Only when both target chains T1 and T2 are present, the walking chain W can completely unblock and expose the entire deoxyribozyme catalytic region, and further hybridize with the track chain Tr on the AuNPs.

The track chain Tr is designed as a double stem-loop structure containing a substrate sequence, and an adenine ribonucleotide (rA) is located near the middle of the larger ring. This DNA secondary structure can enable AuNPs to maintain its stability and dispersed state at high salt concentrations through electrostatic effects (negatively charged phosphate backbones in DNA molecules) and steric repulsion, and the solution color is red. After the walking chain W hybridizes with the track chain Tr, the DNA enzyme will exert its catalytic ability, using Mg ²⁺ as a cofactor to cut the track chain Tr by hydrolyzing the phosphodiester bond between riboadenosine and guanine. Thereafter, the smaller ring and part of the larger ring are separated from the remaining track chain Tr, leaving only truncated single chains on the AuNPs. The walking chain W then hybridizes the next track chain Tr, and a "hybridization-hydrolysis" cycle is carried out, whereby most of the track chain Tr will be cut, and the AuNPs become unstable due to the reduction of the negatively charged main chain and the attenuation of steric repulsion. At this time, at the same salt concentration, AuNPs tend to aggregate, causing the color to change from red to purple, realizing the detection of SARS-CoV-2 target nucleic acid sequences T1 and T2, so that it is possible to judge whether SARS-CoV-2 is contained in the sample to be tested based on the color of the solution.

The beneficial effects of the present invention are as follows:

1. The present invention provides a DNA walker for SARS-CoV-2 detection, which realizes visualized SARS-CoV-2 detection. The DNA walker of the present invention has the advantages of few interference factors, low price and good stability, and the sensitivity can reach 1nM.

2. The detection method provided by the present invention has good selectivity and high sensitivity. In the absence of any sequence, the color of the DNA walker reaction solution will not change, and the target sequence can be distinguished by single nucleotide specificity. When the DNA walker or kit of the present invention is used for detection, no reverse transcription step is required, the use of enzymes, labels or modifications is avoided, the results are visualized, and the need for complex equipment is reduced, providing a convenient, economical, fast and reliable method for SARS-CoV-2 virus detection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the colorimetric detection of nucleic acid fragments by the DNA walker of the present invention.

FIG2 is a native polyacrylamide gel electrophoresis image of different locked chains L1 (A) and L2 (B).

In Figure A: Lane M: 20bp DNA marker; Lanes 1-6 are walking chain W, track chain Tr, locked chain 3bp-L1, 4bp-L1, 5bp-L1 and target chain T1 respectively; Lane 7: walking chain W + track chain Tr; Lane 8: walking chain W + locked chain 3bp-L1; Lane 9: walking chain W + locked chain 4bp-L1; Lane 10: walking chain W + locked chain 5bp-L1; Lane 11: walking chain W/locked chain 3bp-L1 double chain + track chain Tr; Lane 12: walking chain W/locked chain 4bp-L1 double chain + track chain Tr; Lane 13: walking chain W/locked chain 5bp-L1 double chain + track chain Tr; Lane 14: walking chain W/locked chain 3bp-L1 double strand + target chain T1 + track chain Tr; Lane 15: walking chain W/locking chain 4bp-L1 double strand + target chain T1 + track chain Tr; Lane 16: walking chain W/locking chain 5bp-L1 double strand + target chain T1 + track chain Tr.

In Figure B: Lane M: 20bp DNA marker; Lanes 1-6 are walking chain W, track chain Tr, locked chain 5bp-L2, 6bp-L2, 7bp-L2, and target chain T2 respectively; Lane 7: walking chain W + track chain Tr (1:2); Lane 8: walking chain W + locked chain 5bp-L2; Lane 9: walking chain W + locked chain 6bp-L2; Lane 10: walking chain W + locked chain 7bp-L2; Lane 11: walking chain W/locked chain 5bp-L2 double chain + track chain Tr; Lane 12: walking chain W/locked chain 6bp-L2 double chain + track chain Tr; Lane 13: walking chain W/locked chain 7bp-L2 double chain + track chain Tr; Lane 14: walking chain W/locked chain 5bp-L2 double strand + target chain T2 + track chain Tr; Lane 15: walking chain W/locking chain 6bp-L2 double strand + target chain T2 + track chain Tr; Lane 16: walking chain W/locking chain 7bp-L2 double strand + target chain T2 + track chain Tr.

FIG. 3 is a native polyacrylamide gel electrophoresis image illustrating the logical response of locked strands 4 bp-L1 and 6 bp-L2.

In the figure: Lane M: 20bp DNA marker; Lanes 1-6 are walking chain W, track chain Tr, locked chain 4bp-L1, 6bp-L2, target chain T1, T2 respectively; Lane 7: walking chain W + track chain Tr; Lane 8: walking chain W + locked chain L1 + locked chain L2; Lane 9: walking chain W/locked chain L1/locked chain L2 complex + track chain Tr; Lane 10: walking chain W/locked chain L1/locked chain L2 complex + target chain T1; Lane 11: walking chain W/locked chain L1/locked chain L2 complex + target chain T2; Lane 12: walking chain W/locked chain L1/locked chain L2 complex + target chain T1 + track chain Tr; Lane 13: walking chain W/locked chain L1/locked chain L2 complex + target chain T2 + track chain Tr; Lane 14: walking chain W/locked chain L1/ Locking chain L2 complex + target chain T1 + target chain T2 + track chain Tr.

FIG4 shows the UV-visible absorption spectrum of AuNPs (A), a transmission electron microscopy image (B), and a photograph of the AuNP solution (C).

FIG5 is a fluorescence spectrum diagram of the DNA walker of the present invention (A) and a calibration curve of the track chain Tr concentration on the DNA walker and the fluorescence at 520 nm (B).

Figure 6 shows the UV-visible absorption spectrum (A), dynamic light scattering characterization (B), fluorescence spectrum (C), fluorescence intensity change over time (D), and color change of the DNA walker reaction liquid (E) of the DNA walker of the present invention after the reaction when the concentrations of target chain T1 and target chain T2 are both 0 nM and 20 nM.

FIG7 is a diagram showing the color change of a visualized solution of the DNA walker of the present invention when detecting SARS-CoV-2 viruses at gradient concentrations of 0, 2, 4, 8, 12, and 20 nM (A), a diagram showing the relationship between the ultraviolet absorption ratio and the SARS-CoV-2 concentration (B), and a calibration curve of the standard curve (C)

Figure 8 is a visualization diagram of the results of the DNA walker of the present invention in detecting SARS-CoV-2 virus gene fragments without SARS-CoV-2 virus gene fragments, SARS-CoV-2 virus gene fragments with a single target chain T1 or target chain T2, and SARS-CoV-2 virus gene fragments.

In the figure, 0 means that the target chains T1 and T2 are not contained, and 1 means that the target chains T1 or T2 are contained.

Figure 9 is the UV-visible absorption spectrum of the DNA walker of the present invention when detecting SARS-CoV-2 virus gene fragments without SARS-CoV-2 virus gene fragments, SARS-CoV-2 virus gene fragments with a single target chain T1 or target chain T2, and SARS-CoV-2 virus gene fragments.

FIG10 is a diagram (B) showing the visualization observation results of the DNA walker of the present invention when detecting a SARS-CoV-2 viral gene fragment with base mismatch in the target strand T1 or the target strand T2.

In the figure, 0 means that the target chain T1 or target chain T2 is not contained, and 1 means that the target chain T1 or target chain T2 is contained.

Detailed ways

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Reagents and materials:

DNA sequences (Table S1) were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China).

40% acrylamide/bisacrylamide (19:1) solution, ammonium persulfate (APS), N,N,N′,N′-tetramethylethylenediamine (TEMED), Tris, and tetrasodium ethylenediaminetetraacetate (EDTA) were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China).

SYBR Gold nucleic acid gel stain was purchased from Thermo Fisher Scientific.

Hydrogen tetrachloroaurate (III) trihydrate (HAuCl ₄ ·3H ₂ O) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (Missouri, USA).

All other reagents were of analytical grade and used as received. Ultrapure water (18.25 MΩ·cm) obtained from a UP water purification system was used throughout the experiments.

The 20 bp DNA ladder was purchased from Bio-Ray Biomedical Technology Co., Ltd. (Beijing).

UV-visible absorption spectra were recorded using a TU-1901 spectrometer (Puxi, China). Fluorescence emission spectra were measured using a F-320 spectrofluorometer (Tianjin Gangdong Technology Development Co., Ltd., China). Stained polyacrylamide gels were imaged on a GelDocTM XR ⁺ imaging system (Bio-RAD Laboratories Inc., USA). Dynamic light scattering (DLS) measurements were performed on a nanoparticle size potentiometer (Malvern, UK). Transmission electron microscopy (TEM) measurements were performed on a JSM-6700F transmission electron microscope (JEOL, Japan) with an accelerating voltage of 200 kV. All other products were commercially available unless otherwise specified.

Table 1. Oligonucleotide sequences used in the examples

In Table 1, bold letters represent the binding arms on both sides of the catalytic core and the corresponding complementary bases; italic letters represent the catalytic core; underlined letters represent mismatched bases; and the number of nbp-L represents the number of bases in the locking chain binding arm. The concentration of the oligonucleotide was determined based on the UV absorbance at 260 nm and the extinction coefficient of the sequence. The locking chain consists of a target recognition domain and a blocking domain with different lengths.

Example 1

According to the SARS-CoV-2 viral genome information (NCBI reference sequence: NC_045512.2) disclosed in the NCBI database, the present invention selects two RNA gene fragments ORF1ab and N as target strands T1 and T2 from the open reading frame and nucleocapsid, and then designs walking strand W, 3bp-locking strand L1, 4bp-locking strand L1, 5bp-locking strand L1, 5bp-locking strand L2, 6bp-locking strand L2, 7bp-locking strand L2, and track strand Tr according to the target strands T1 and T2, and the specific sequences are shown in Table 1. Then it was synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China).

The locking strands consist of a target recognition domain and a closing domain of varying lengths.

Example 2

1. Preparation and storage of DNA and RNA solutions

Concentrated DNA stock solutions of the sequences described in Table 1 were prepared in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and diluted to working concentrations with HEPES buffer (10 mM HEPES, 300 mM NaCl, pH 7.0).

Concentration method: Dissolve the nucleic acid sequence in the TE buffer solution in the microliters indicated on the tube, and determine the concentration of the concentrate by ultraviolet absorption spectroscopy.

2. Selection of locking chain L1 and locking chain L2

1.44 μL of 10 mM walking chain W and 1.50 μL of 10 mM locking chain (nbp-L1, nbp-L2) were mixed in annealing buffer (25 mM Tris-acetate, 200 mM NaCl, pH 8.0) to a total volume of 26.12 μL. The mixture was heated to 95°C for 2 min and then cooled to 25°C to obtain an annealing mixture.

2.88 μL of 10 mM track chain Tr and 1 μL of 600 mM MgCl ₂ were added to the annealing mixture, incubated at 25°C for 10 min, and the incubation product was subjected to polyacrylamide gel electrophoresis. In order to verify the displacement of the target sequence to the locked chain, 1.38 μL of DEPC-H ₂ O, 26.12 μL of the annealing mixture and 1.5 μL of 10 mM target chains T1 and T2 were mixed evenly, incubated at 25°C for 30 min, and the incubation product and other products in this reaction were subjected to polyacrylamide gel electrophoresis. The results are shown in Figure 2.

As shown in Figure 2, the length of the locked strand is crucial for the inactivation of the DNAzyme in the absence of a target and its activation in the presence of a target.

Lanes 8-10 show that compared with the walking chain W, track chain Tr, locked chain 3bp-L1, 4bp-L1, 5bp-L1, and target chain T1 fragments in lanes 1 to 6, after incubation of the walking chain W and the locked chain, the three locked chains can hybridize with the walking chain W, but the addition of the track chain Tr can produce competitive hybridization with the locked chain for the walking chain W.

Lanes 11-13 show that compared with the locked chains 4bp-L1 and 5bp-L1, the locked chain 3bp-L1 cannot block the walking chain W well, and some track chains Tr can still compete with the walking chain W for hybridization and be cut. The intensity of the track chain Tr band in lane 11 is reduced, and a faint band with the same mobility as the cut product in lane 7 appears below. Insufficient blocking of the walking chain W will cause the DNA walker to produce nonspecific responses even when the target chain T1 is absent, which will lead to high background or even false positive signals after triggering amplification, so only 4bp-L1 and 5bp-L1 meet the requirements.

Lanes 14-16 show that the presence of target chain T1 should displace the locked chain and leave the DNA enzyme single chain. The tests of 3bp-L1, 4bp-L1 and 5bp-L1 show that the band corresponding to the W-L1 complex gradually appears, while the intensity of the band corresponding to the walking chain W gradually decreases. Since there are more base pairings between the walking chain W and the locked chain 5bp-L1, the duplex formed is more stable, resulting in limited displacement efficiency of the target chain T1, and this low unblocking efficiency will reduce the sensitivity of the walker. It can be realized that slight changes in base pairs will have a huge impact on the interaction between DNA chains. The low background and high specificity when the target chain T1 is absent and the high unblocking efficiency when the target chain T1 is present are the criteria for judgment, and the locked chain is determined to be 4bp-L1. Similarly, 6bp-L2 can still effectively block the walking chain in the presence of the track chain Tr, and can also effectively displace it after adding the target target chain T2 nucleic acid fragment, and the locked chain is determined to be 6bp-L2.

3. Logical response of locked strands 4bp-L1 and 6bp-L2

1.44 μL of 10 mM walking chain W, 1.50 μL of 10 mM locking chain 4 bp-L1 and 1.50 μL of 10 mM locking chain 6 bp-L2 were mixed in 23.12 μL of annealing buffer for annealing to obtain a walking chain W/locking chain L1/locking chain L2 complex. Then 3 μL of 5 mM target chain T1, 3 μL of 5 mM target chain T2 or 3 μL of 5 mM target chains T1 and T2 were added to 2.88 μL of 10 mM track chain Tr and 23.12 μL of walking chain W/locking chain L1/locking chain L2 complex, respectively, and incubated at 25°C for 30 minutes, and then 1 μL of 600 mM MgCl ₂ was added and incubated for 10 minutes. The incubation product and other products in this reaction were subjected to polyacrylamide gel electrophoresis, and the results are shown in Figure 3.

As can be seen from Figure 3, lane 7 shows that when the walking chain W and the track chain Tr are incubated with Mg ²⁺ , the track chain Tr is cut into two short oligonucleotides (Tr1 and Tr2) with faster migration speed due to the hydrolysis of the substrate sequence by the DNAzyme.

Lanes 8-9 show that the walking strand W has been completely blocked by the locking strands 4bp-L1 and 6bp-L2, because the track strand Tr will not be cut even if Mg ²⁺ is introduced.

Lane 10 shows that the target strand T1 specifically displaces the locked strand L1 from the walking strand W/locked strand L1/locked strand L2 complex, forming a walking strand W-locked strand L2 double strand and a locked strand L1-target strand T1 double strand.

Lane 11 shows that the target strand T2 specifically displaces the locked strand L2 from the walking strand W/locked strand L1/locked strand L2 complex to form a walking strand W-locked strand L1 double strand and a locked strand L2-target strand T2 double strand.

Lanes 12-13 show that no matter whether the locked chain L1 is unblocked by the target chain T1 alone, or the locked chain L2 is unblocked by the target chain T2 alone, the track chain Tr will not be cut by the walking chain W. In these cases, since the other hybridization arm of the walking chain W is blocked, the deoxyribozyme is still in an inactive state. Only when both the target chains L1 and L2 are present can the walking chain W be completely unblocked. Then the deoxyribozyme regains its activity and cuts the track chain Tr into two shorter fragments, Tr1 and Tr2. Therefore, the doubly blocked walking chain W can only bind to the track chain Tr in the presence of both the target chains L1 and L2, and then the deoxyribozyme hydrolyzes the substrate to realize an "AND" logic gate.

In summary, the walking chain W, locking chain L1, locking chain L2 and track chain Tr designed in the present invention ensure that the deoxyribozyme remains inactive when only one of the target chains T1 or T2 is present, and can only be unblocked in the presence of SARS-CoV-2 containing both target chains T1 and T2 to catalyze the hydrolysis of the substrate.

Example 3

1. The preparation method of gold nanoparticles is as follows:

Prepare 10 mL of a 38.8 mM sodium citrate aqueous solution and 100 mL of a 1 mM HAuCl ₄ aqueous solution; heat the HAuCl ₄ aqueous solution to boiling, then quickly add the sodium citrate aqueous solution to the HAuCl ₄ aqueous solution while vigorously stirring, react for 10 minutes under heating and boiling, stir for 15 minutes after the reaction is completed, cool to 25°C, and filter through a 0.22 μm filter to obtain gold nanoparticles (AuNPs).

A drop of gold nanoparticle solution was added to the carbon-coated copper grid and dried at room temperature to prepare the sample for TEM characterization. Characterization was performed on a JSM-6700F transmission electron microscope with an accelerating voltage of 200 kV.

The UV-visible absorption spectrum of the AuNPs in this example is shown in FIG4A , the transmission electron microscope (TEM) image is shown in FIG4B , and the photograph of the AuNP solution is shown in FIG4C .

As shown in Figures 4A-C, AuNPs were successfully synthesized, showing a maximum absorbance at 520 nm, appearing red, and having a uniform size distribution with an average size of 13 nm.

2. A method for preparing a DNA walker for SARS-CoV-2 detection, comprising the following steps:

(1) The walking strand W, the locking strand 4bp-L1 and the locking strand 6bp-L2 were mixed in an annealing buffer (25 mM Tris-acetic acid, 200 mM NaCl, pH 8.0) at a molar ratio of 1:3:3, and then heated at 95°C for 10 min and cooled to 25°C to obtain a completely closed walking strand; the completely closed walking strand and TCEP were then mixed at a molar ratio of 1:50 and incubated for 2 h to obtain a thiol-completely closed walking strand;

(2) heating the track chain Tr at 95°C for 2 min to obtain an annealed track chain Tr;

(3) AuNPs, thiol-completely closed walking chains and annealed track chains Tr were mixed evenly in a molar ratio of 1:20:200, and then incubated at 4°C in the dark for 16 h. Then, NaCl solution was added at intervals of 40 min to make the final NaCl concentration 0.2 M. The mixture was incubated at 4°C in the dark for 24 h. The precipitate was obtained by centrifugation at 4°C, 14000 rpm for 30 min. The precipitate was washed three times with washing solution (25 mM Tris-acetate, pH 8.0) to obtain a DNA walker (W/Tr/AuNP DNA walker) for SARS-CoV-2 detection. Finally, the DNA walker was stored at a concentration of 60-80 nM in storage solution (25 mM Tris-acetate, 100 mM NaCl, pH 8.0) at 4°C in the dark.

The number and concentration of track chain Tr on the W/Tr/AuNP DNA walker were determined by the DTT displacement method, and then a standard calibration curve was drawn according to the concentration of the track chain Tr labeled with the fluorophore. The results are shown in Figure 5.

As shown in Figure 5 , there are 23 hairpin track chains conjugated on the AuNP of the W/Tr/AuNP DNA walker.

Example 4

The DNA walker prepared in Example 3 and the target strands T1+T2 were reacted in a reaction buffer (25 mM Tris-acetate, 225 mM NaCl, 20 mM MgCl ₂ , pH 8.0) at 25°C for 3 h, and then CaCl ₂ was added, with the final concentration of CaCl ₂ in the buffer being 180 mM, and visual observation was performed. The reaction was divided into two groups. In group 1, the concentration of the DNA walker was 3 nM, and the concentrations of the target strands T1 and T2 were both 0 nM; in group 2, the concentration of the DNA walker was 3 nM, and the concentrations of the target strands T1 and T2 were both 20 nM. The results are shown in FIG6

As shown in Figure 6E, when the W/Tr/AuNP DNA walker detects the target chain T1 and the target chain T2, the color of the solution can be visually observed to change from red to purple. As shown in Figure 6A, compared with the case of target chain T1 and target chain T2, the maximum absorbance is red-shifted, and a new absorption band near 620nm appears. The spectral change explains the color change and indicates the aggregation of the three-dimensional DNA walker. As shown in Figure 6B, in the presence of both target chain T1 and target chain T2, the average hydration diameter of the DNA walker as a whole increases from 68nm to 342nm. As shown in Figure 6C, in the presence of both target chain T1 and target chain T2, the fluorescence increases significantly, which is the result of the walking chain W being unblocked by the target chain T1 and target chain T2 and walking and cutting the track chain on the AuNP surface. As shown in Figure 6D, the rate at which the walking chain W cuts the hairpin track chain Tr is higher than its cutting on the straight track chain.

Example 5

1. SARS-CoV-2 virus gene fragments with gradient concentrations of 0, 2, 4, 8, 12, and 20 nM were used as test samples, and detection and UV absorption spectrum measurement were performed according to the method described in Example 4. The results are shown in Figure 7.

As shown in Figure 7, as the concentration of SARS-CoV-2 virus gene fragments increases, the color of the W/Tr/AuNP DNA walker reaction liquid of the present invention gradually changes from red to purple, and then to purple-gray, realizing the visual detection of SARS-CoV-2. As the concentration of SARS-CoV-2 virus gene fragments increases from 0 to 10nM, the absorbance ratio gradually increases, and then reaches saturation until 20nM; within the concentration range of 0 to 10nM, the value of A620/A520 has a good linear relationship with the concentration of SARS-CoV-2 virus gene fragments, and the detection limit of the W/Tr/AuNP DNA walker of the present invention is 1nM.

2. Using a SARS-CoV-2 virus gene fragment without SARS-CoV-2 (SARS-CoV-2 virus concentration of 0 nM), a SARS-CoV-2 virus gene fragment containing only the target chain T1 (concentration of 20 nM), a SARS-CoV-2 virus gene fragment containing only the target chain T2 (concentration of 20 nM) and a SARS-CoV-2 virus gene fragment (concentration of 20 nM) as samples to be tested, detection and ultraviolet absorption spectrum measurement were performed according to the method described in Example 5, and the results are shown in Figures 8 and 9.

As can be seen from Figures 8 and 9, the W/Tr/AuNP DNA walker of the present invention is specific to the SARS-CoV-2 virus gene fragment containing target chains T1+T2. When there are only target chains T1+T2, the reaction liquid shows purple, and when there are no target chains T1 and T2, or only target chains T1 or T2, they all show red.

3. The SARS-CoV-2 virus gene fragment with a 1bp base mismatch in the target chain T1 (1bp-Mismatch-T1, Table 1, concentration of 20nM), the SARS-CoV-2 virus gene fragment with a 2bp base mismatch in the target chain T1 (2bp-Mismatch-T1, Table 1, concentration of 20nM), the SARS-CoV-2 virus gene fragment with a 3bp base mismatch in the target chain T1 (3bp-Mismatch-T1, Table 1, concentration of 20nM), the SARS-CoV-2 virus gene fragment with a 1bp base mismatch in the target chain T2 (1bp-Mismatch-T2, Table 1, concentration of 20nM), the SARS-CoV-2 virus gene fragment with a 2bp base mismatch in the target chain T2 (2bp-Mismatch-T2, Table 1, concentration of 20nM), the target chain T2 The SARS-CoV-2 virus gene fragment with a 3bp base mismatch (3bp-Mismatch-T1, Table 1, concentration of 20 nM) and the SARS-CoV-2 virus gene fragment (concentration of 20 nM) were used as test samples and detected according to the method described in Example 5. The results are shown in Figure 10.

As shown in Figure 10, the SARS-CoV-2 virus gene fragments with base mismatches in the sequences of the target chains T1 or T2 also appear red, indicating that base mismatches in a single target chain T1 or T2 will not cause the W/Tr/AuNP DNA walker to change color, thereby increasing the detection accuracy and ensuring the detection reliability of the W/Tr/AuNP DNA walker.

Claims (10)

1. A DNA walker for SARS-CoV-2 detection, characterized in that the DNA walker comprises a walking chain W, a locking chain L1, a locking chain L2, a track chain Tr, a target chain T1, a target chain T2 and nanogold particles; The nucleotide sequence of the walking strand W is: 5'-HS-(T) ₄₂ TGGTTCAATCTGTCAATCTCTTCTCCGAGCCGGTCGAAATAGTCCATAACCTTTCCACA-3'; The nucleotide sequence of the locked strand L1 is: 5'-TGCGGTATGTGGAAAGGTTATGGACTA-3'; The nucleotide sequence of the locked strand L2 is: 5'-AAGAGATTGACAGATTGAACCAGCTTGAG-3'; The nucleotide sequence of the track chain Tr is: 5'-HS-(T) ₆ GGCTGTGGACTAT/rA/GGAAGAGATTCAGCCCGCGTTTTTTTCGCG-6-Carboxyfluorescein-3'; The nucleotide sequence of the target strand T1 is: 5'-CCATAACCTTTCCACATACCGCA-3'; The nucleotide sequence of the target chain T2 is: 5'-CTCAAGCTGTTCAATCTGTCAA-3'. 2. The DNA walker for SARS-CoV-2 detection according to claim 1, characterized in that the preparation method of the gold nanoparticles is as follows: A sodium citrate aqueous solution with a concentration of 38.8 mM and a HAuCl ₄ aqueous solution with a concentration of 1 mM were prepared; the HAuCl ₄ aqueous solution was heated to boiling, and then the sodium citrate aqueous solution was added to the HAuCl ₄ aqueous solution while stirring, and the mixture was reacted for 10 minutes under the heating boiling state. After the reaction was completed, the mixture was stirred for another 15 minutes, cooled to 25° C., and filtered to obtain gold nanoparticles. 3. A SARS-CoV-2 detection kit, characterized in that the kit comprises the DNA walker according to claim 1 and _MgCl2 solution. 4. The detection kit according to claim 3, characterized in that the working concentration of the DNA walker in the kit is 3 nM, and the working concentration of the _MgCl2 solution is 180 mM. 5. The method for preparing the DNA walker for SARS-CoV-2 detection according to claim 1, characterized in that it comprises the following steps: (1) The walking chain W, the locking chain L1 and the locking chain L2 were mixed evenly in the annealing buffer, then heated at 95°C for 10 min, and cooled to 20-30°C to obtain a completely closed walking chain; the completely closed walking chain was then incubated with TCEP for 2 h to obtain a thiol-completely closed walking chain; (2) heating the track chain Tr at 95°C for 10 min to obtain the annealed track chain Tr; (3) AuNPs, thiol-completely closed walking chains and annealed track chains Tr were mixed evenly, and then incubated at 4°C in the dark for 16 h. Then, NaCl solution was added at intervals of 40 min until the final NaCl concentration was 0.2 M. The mixture was incubated at 4°C in the dark for 24 h. After centrifugation and washing, the DNA walker for SARS-CoV-2 detection was obtained. 6 . The preparation method according to claim 5 , characterized in that in step (1), the molar ratio of the walking chain W, the locking chain L1 and the locking chain L2 is 1:3:3. 7. The preparation method according to claim 5, characterized in that in step (1), the molar ratio of the completely closed walking chain to TCEP is 1:50. 8. The preparation method according to claim 5, characterized in that in step (3), the molar ratio of the AuNPs, the thiolated completely closed walking chain and the annealed track chain Tr is 1:20:200. 9. A method for detecting SARS-CoV-2 for non-diagnostic purposes using the DNA walker of claim 1, characterized in that it comprises the following steps: The sample to be tested is added to a buffer solution with a final concentration of 3 nM of DNA Walker, reacted at 20-30°C for 2.5-3.5 hours, and then CaCl ₂ is added. When the incubation solution turns purple, the sample to be tested contains SARS-CoV-2. 10. The method for detecting SARS-CoV-2 for non-diagnostic purposes according to claim 9, wherein the final concentration of _CaCl2 in the buffer is 180 mM.