Dual-signal biosensor constructed based on AUW-SDA technology and application of dual-signal biosensor in nucleic acid detection of new coronavirus

Abstract

The invention discloses a dual-signal biosensor constructed based on an AUW-SDA technology and application of the dual-signal biosensor in nucleic acid detection of new coronavirus. Compared with a biosensor constructed by using a traditional SDA technology, the dual-signal biosensor constructed based on the AUW-SDA technology realizes direct detection of a breakpoint-free long gene sequence, target replacement and reutilization and output of multiple editable signal sequences. Based on a fluorescence signal, a new coronavirus target sequence with the minimum of 0.679 fM can be detected; based on a chemical chromogenic signal, a new coronavirus target sequence with a minimum of 13.5 fM can be detected. The nucleotide probe used in the technology does not need to be modified, the cost is low, nucleic acid detection biosensors of various signal output types can be adapted only by replacing and replacing primer sequences, and the method has wide application prospects in medical detection and biological research.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a dual-signal biosensor constructed based on AUW-SDA technology and its application in the detection of new coronavirus nucleic acid. Background Art

[0002] Strand displacement amplification (SDA) technology can effectively achieve efficient amplification of nucleic acid targets under constant temperature conditions, and its probe design is simple, making it an important isothermal amplification technology. Currently, SDA technology can achieve efficient amplification of targets such as genomic DNA and viral RNA. At the same time, SDA technology is also widely used in the construction of biosensors. As a method of signal transduction and amplification, it can be adapted to biosensors with various types of signal outputs.

[0003] There are still some problems in the application of traditional SDA technology to the construction of nucleic acid detection biosensors: inability to detect long-chain nucleic acid targets (depending on the free 3' end of the target nucleic acid), low sequence freedom of amplified products (containing the detection target sequence and thus difficult to adapt to downstream signal output), low efficiency of target nucleic acid molecule utilization (unable to achieve replacement and reuse of target molecules), etc. Through improvements by some researchers, some SDA modifications can solve some of these problems: by designing single-molecule allosteric probes to break away from the dependence on the free 3' end of the nucleic acid target to detect long-chain nucleic acid targets; by extending the template to amplify any product; by using auxiliary primers to achieve replacement and reuse of the target. However, there is still a lack of SDA technology that can completely unify these functions and can be applied to nucleic acid biosensor detection. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a dual-signal biosensor based on AUW-SDA technology, which includes:

[0007] A sequence probe, wherein the sequence probe comprises a first hybridization region and a second hybridization region, wherein the first hybridization region and the second hybridization region are connected by a loop region to form a hairpin structure; the first hybridization region contains a sequence complementary to a target, and the target binds to the sequence of the first hybridization region to open the hairpin structure; the sequence of the loop region contains a nicking endonuclease site;

[0008] A displacement primer, wherein the displacement primer contains a sequence complementary to the second hybridization region and a sequence complementary to the product strand, and the displacement primer binds to the sequence probe to form a first 3' end that can be recognized and paired by a KF polymerase lacking 3'→5' exonuclease activity;

[0009] A nicking endonuclease for cleaving the sequence probe to form a second 3' end that can be recognized and paired by a KF polymerase lacking 3'→5' exonuclease activity;

[0010] The KF polymerase lacking 3'→5' exonuclease activity pairs with the first 3' end to initiate polymerization, and uses the sequence probe as a template to synthesize a corresponding complementary sequence to synthesize a sequence probe-displacement primer complex; the KF polymerase lacking 3'→5' exonuclease activity pairs with the second 3' end to initiate polymerization, and uses the displacement primer as a template to synthesize a corresponding product sequence;

[0011] Wherein, the nicking endonuclease cuts the sequence probe to generate a product sequence.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology, characterized in that: comprising:

[0013] When the sequence probe does not recognize the target, the hairpin structure is maintained, and the displacement primer has difficulty in invading the sequence probe in the hairpin configuration to form the first 3' end of the pair, and it cannot be recognized by the KF polymerase lacking 3'→5' nuclease activity;

[0014] When the sequence probe recognizes the target, the target binds to the sequence probe, the sequence probe opens the hairpin structure, and the displacement primer binds to the sequence probe to form a paired first 3' end that can be recognized by the KF polymerase lacking 3'→5' exonuclease activity;

[0015] KF polymerase lacking 3'→5' exonuclease activity initiates polymerization to complete the complementary sequence at the nicking enzyme site on the sequence probe. At the same time, the corresponding complementary sequence is synthesized using the sequence probe as a template, displacing the target already bound to the sequence probe, and synthesizing a sequence probe-displacement primer complex with a complete nicking enzyme site. The displaced target enters the next cycle;

[0016] The sequence probe-displacement primer complex makes a cut at the nicking endonuclease site under the action of the nicking endonuclease, and the second 3' end that pairs with the KF polymerase lacking 3'→5' nuclease activity is formed at the cut;

[0017] Under the action of KF polymerase lacking 3'→5' exonuclease activity, a strand displacement amplification reaction occurs at the incision, and the corresponding product sequence is synthesized using the displacement primer as a template;

[0018] The product sequence depends on the sequence on the displacement primer that is complementary to the product chain and is completely independent of the target sequence. By designing the sequence on the displacement primer that is complementary to the product chain, any product sequence can be amplified to adapt to a variety of nucleic acid-based biosensors to detect target concentrations.

[0019] As a preferred embodiment of the method for detecting target concentration of the dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the sequence complementary to the product chain on the displacement primer is designed as the complementary sequence of the CRISPR / Cas12a system activator, which is recorded as RP-CRISPR / Cas, and the CRISPR / Cas12a system activator is generated by chain displacement amplification reaction, activating the Cas12a protein to make it have trans-nicking activity and cut the FQ probe. After the FQ probe is cut, the BHQ1 quenching group on the FQ probe is separated from the 6-FAM fluorescent group, and under the irradiation of 494nm blue light, an excitation light with a peak of 519nm is emitted to achieve fluorescence signal output, which is the CRISPR / Cas12a fluorescence signal characterization system;

[0020] As a preferred embodiment of the method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the sequence on the displacement primer complementary to the product chain is designed as a complementary sequence of the G-quadruplex, denoted as RP-G4 / Hemin, and a sequence carrying the G-quadruplex is generated through a chain displacement amplification reaction. After forming a G4 / Hemin complex with Hemin, the sequence catalyzes the decomposition of hydrogen peroxide and oxidizes TMB. The oxidized TMB changes from colorless to blue. After adding concentrated acid, the oxidation process is stopped, and the TMB appears as yellow with an absorption peak at 450nm, thereby realizing a visualized signal output, i.e., a G4 / Hemin-TMB chemical color development signal characterization system.

[0021] As a preferred embodiment of the method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the sequence probe corresponds to a nucleic acid sequence as shown in one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.7-10, and SEQ ID No.19-23.

[0022] As a preferred embodiment of the method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the nucleic acid sequence corresponding to the replacement primer RP-CRISPR / Cas is shown in SEQ ID No. 3, and the nucleic acid sequence corresponding to the FQ probe is shown in SEQ ID No. 5.

[0023] As a preferred embodiment of the method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the nucleic acid sequence corresponding to the replacement primer RP-G4 / Hemin is shown in one of SEQ ID No.4 and SEQ ID No.11-18.

[0024] As a preferred embodiment of the method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, when the biosensor based on the AUW-SDA-CRISPR / Cas12a fluorescence characterization system is used for the detection of new coronavirus nucleic acid, the new coronavirus nucleic acid is used as the target, and different concentrations of new coronavirus nucleic acid virus sequence standard solutions are mixed with the AUW-SDA reaction solution in CutSmart buffer for reaction, and then the CRISPR / Cas12a reaction solution is added, and the FQ probe is added, the reaction is mixed, the excitation light intensity is detected, a standard curve is drawn, and the concentration is determined.

[0025] As a preferred embodiment of the method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, when the biosensor based on the AUW-SDA-G4 / Hemin chemical color development characterization system is used for the detection of new coronavirus nucleic acid, different concentrations of new coronavirus nucleic acid virus sequence standard solutions are mixed with the AUW-SDA reaction solution in CutSmart buffer for reaction, and then Hemin is added and mixed in the AUW-SDA-G4 buffer; then TMB and hydrogen peroxide solution are added and mixed in the AUW-SDA-TMB color development buffer to carry out a color development reaction, the absorbance of the reaction solution is detected to draw a standard curve and determine the concentration.

[0026] As a preferred embodiment of the method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology of the present invention, the components of the AUW-SDA reaction solution are 10-100 nM sequence probe, 10-100 nM replacement primer, 0.5-2.5 mM dNTP, 0.005-0.1 U / μL Nt.BbvCI nicking endonuclease and 0.005-0.02 U / μL KF polymerase lacking 3'→5' exonuclease activity, and the solution system is CutSmart buffer;

[0027] Among them, the Nt.BbvCI nicking endonuclease can be replaced by other nicking endonucleases with the same effect; the CutSmart buffer composition is 50mM potassium acetate, 20mM Tris-Acetate, 10mM magnesium acetate and 100μg / mL BSA, pH7.9; the AUW-SDA reaction solution is reacted at 37°C for 1 to 4h, and the reaction is terminated by incubation at 70 to 95°C for 5 to 25min.

[0028] As a preferred embodiment of the method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology described in the present invention, the AUW-SDA-G4 buffer component is 10-20mM HEPES, 50-400mM ammonium chloride, 0.0125-0.05% Triton X-100 and 0.5-1% dimethyl sulfoxide; the AUW-SDA-TMB color development buffer component is 26.2mM citric acid, 51.4mM disodium hydrogen phosphate dodecahydrate, 10-50mM potassium chloride and 50-400mM ammonium chloride.

[0029] Beneficial effects of the present invention:

[0030] (1) AUW-SDA technology can achieve amplification activation without relying on the 3' end of the target, and can be used to detect long nucleic acid sequences; it can achieve target replacement to obtain higher sensitivity and detection efficiency; it can achieve arbitrary signal output sequence amplification by designing variable sequences on the replacement primer RP sequence, so as to adapt to nucleic acid detection biosensors with various signal output types. This technology has been adapted to CRISPR / Cas12a nucleic acid detection biosensors and G4 / Hemin-TMB nucleic acid detection biosensors.

[0031] (2) AUW-SDA solves the main shortcomings of traditional SDA technology in biosensors. The output nucleic acid signal has a wide linear range and can be used for efficient detection of various types of nucleic acid targets.

[0032] (3) The development cost is low. Only by replacing the sequence probe and the replacement primer, it can be used to detect different nucleic acid targets; and only by changing the replacement primer, it can adapt to different types of nucleic acid detection biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0034] Figure 1This is a technical schematic diagram of the dual-signal biosensor constructed based on AUW-SDA technology in Example 1 of the present invention for detecting the nucleic acid sequence of the new coronavirus.

[0035] Figure 2 This is a feasibility analysis diagram of the biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system in Example 2 and Comparative Example 1 of the present invention.

[0036] Figure 3 AUW-SDA-CRISPR / Cas12a detects novel coronavirus nucleic acid fragments and standard curves in Example 3 of the present invention.

[0037] Figure 4 This is a feasibility analysis diagram of the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system in Example 4 and Comparative Example 2 of the present invention.

[0038] Figure 5 This is a condition optimization diagram of the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system in Example 5 of the present invention.

[0039] Figure 6 AUW-SDA-G4 / Hemin in Example 6 of the present invention detects the nucleic acid fragment and standard curve of the new coronavirus.

[0040] Figure 7 It is an optimization diagram (A) and a linear diagram (B) of the biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system in Example 7 of the present invention for detecting the novel coronavirus nucleic acid N gene, and a comparison diagram (C) of the RT-qPCR effect between the biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system in Example 7 and the comparative example 3.

[0041] Figure 8 The optimization diagram (A) and linear diagram (B) of the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system in Example 7 of the present invention for detecting the novel coronavirus nucleic acid N gene, and the comparison diagram (C) of the RT-qPCR effect between the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system in Example 7 and the comparative example 3 are shown.

[0042] Fig. 9 This is a specific graph of the detection of novel coronavirus fragments by the biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system in Comparative Example 4 of the present invention.

[0043] Fig.10This is a specific diagram of the biosensor based on the AUW-SDA-G4 / Hemin high-efficiency chemical colorimetric characterization system in comparative example 5 of the present invention for detecting the novel coronavirus fragment. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0047] SDA: Strand displacement amplification, an enzymatic DNA in vitro isothermal amplification method. Nicking endonuclease recognition sequences are added to both ends of the target DNA. The nicking endonuclease opens a gap in the double-stranded DNA at its recognition site, and the DNA polymerase then extends the 3' end of the gap and replaces the next DNA chain. The replaced single-stranded DNA can bind to the primer and be extended into a double-stranded DNA by the DNA polymerase. This process is repeated continuously, so that the target sequence is amplified efficiently.

[0048] AUW-SDA (All-U-Want-Strand Displacement Amplification) is a new technology based on the extended design of SDA technology, and is used in the present invention to detect the nucleic acid of the new coronavirus.

[0049] CRISPR / Cas12a: A ribonucleoprotein complex that has cis- and trans-deoxyribonuclease activity after binding to an activation sequence and is used in the field of gene editing or gene testing.

[0050] Hemin: Hemin.

[0051] G-quadruplex: A nucleic acid secondary structure formed by the folding of nucleic acids rich in tandem repeats of guanine (G).

[0052] Tris-HCl: tris(hydroxymethyl)aminomethane hydrochloride.

[0053] Tris-Acetate: Tris(hydroxymethyl)aminomethane acetate.

[0054] HEPES: N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) sodium salt.

[0055] Triton X-100: Triton X-100.

[0056] TMB: 3,3',5,5'-tetramethylbenzidine.

[0057] dNTP: deoxyribonucleoside triphosphate.

[0058] BSA: bovine serum albumin.

[0059] Double distilled water: a type of heavily distilled water, which is obtained by distilling water again after it has been distilled once.

[0060] Example 1

[0061] This embodiment provides a method for constructing a dual-signal biosensor based on AUW-SDA technology, specifically:

[0062] The dual-signal biosensor constructed by AUW-SDA technology includes the sequence probe SP, replacement primer RP, Nt.BbvCI nicking endonuclease and KF polymerase (3'→5'Exo-) that constitute AUW-SDA; the CRISPR / Cas12a high-efficiency fluorescence characterization system and the G4 / Hemin-TMB high-efficiency chemical colorimetric characterization system that constitute the characterization elements.

[0063] The replacement primer RP was diluted to 1 μM with double distilled water and stored at 4°C until use.

[0064] The sequence probe SP was diluted in Tris-Na buffer to 1 μM, heated at 95°C for 5 minutes, then slowly cooled to 25°C at -1°C / min, and stored at 4°C for future use; the formula of Tris-Na buffer was: 20 mM Tris-HCl, 400 mM sodium chloride, pH 7.4.

[0065] The oligonucleotide sequences involved in the present invention are shown in Table 1. All sequences have been named according to the standard format, SP-loop length-leg length, RP-signal output mode-primer region length-leg length. Among them, crRNA and SARS-CoV-2 are ribonucleic acid (RNA), and the remaining sequences are deoxyribonucleic acid (DNA).

[0066] Table 1 Oligonucleotide sequences

[0067]

[0068]

[0069]

[0070] The reagents used in the embodiments of the present invention are commercially available unless otherwise specified, as shown in Table 2. The ribonucleic acid (RNA) used in the present invention was purchased from Suzhou Junji Biology; the deoxyribonucleic acid (DNA) was purchased from Shanghai Shenggong Biology. The LbCas12a protein was obtained by prokaryotic induced expression and purification. For specific methods, see: Analytica Chimica Acta, 2023, 1283: 341950.

[0071] Table 2 Reagent names and sources

[0072] name Commercial channels Part Number Tris-HCl Shanghai Bioengineering A610103 HEPES Shanghai Bioengineering A600265 dNTP Shanghai Bioengineering A610056 Hemin Shanghai Bioengineering A419195 TMB Shanghai Bioengineering A600954 Sodium chloride Shanghai Bioengineering A610476 Potassium chloride Shanghai Bioengineering A610440 Ammonium chloride Shanghai Bioengineering A600034 Magnesium chloride Shanghai Bioengineering A610328 TritonX-100 Shanghai Bioengineering A417820 BSA Aladdin B265994 Dimethyl sulfoxide Shanghai Bioengineering A460700 Disodium hydrogen phosphate dodecahydrate Shanghai Bioengineering A501725 Citric Acid Shanghai Bioengineering A501702 Magnesium acetate Aladdin M108884 Potassium acetate Aladdin P108329 Nt.BbvCI nicking endonuclease NewEnglandBiolabs B7204 KF polymerase (3'→5'Exo-) NewEnglandBiolabs R0632

[0073] The biosensor based on the CRISPR / Cas12a efficient fluorescence characterization system takes different concentrations of the new coronavirus nucleic acid sequence standard solution and mixes it with the AUW-SDA reaction solution in CutSmart buffer for reaction, then adds the CRISPR / Cas12a reaction solution and the FQ probe, mixes the reaction, detects the excitation light intensity, draws a standard curve, and determines the concentration.

[0074] The biosensor based on the G4 / Hemin-TMB efficient chemical colorimetric characterization system takes different concentrations of the new coronavirus nucleic acid sequence standard solution and mixes it with the AUW-SDA reaction solution in the CutSmart buffer for reaction. Then, Hemin is added and mixed in the AUW-SDA-G4 buffer. Then, AUW-SDA-TMB colorimetric buffer, TMB, and hydrogen peroxide solution are added and mixed in the AUW-SDA-TMB colorimetric buffer for chemical colorimetric reaction. The absorbance of the reaction solution is detected to draw a standard curve and determine the concentration.

[0075] The principle of the dual-signal biosensor based on AUW-SDA technology to detect the nucleic acid sequence of the new coronavirus is as follows Figure 1 As shown, the complete sensor includes the recognition and signal transduction part based on AUW-SDA, and two optional CRISPR / Cas12a fluorescence and G4 / Hemin-TMB chemical colorimetric signal characterization parts. The target site on the new coronavirus sequence is identified by the sequence probe SP, and under the action of the replacement primer RP and the enzyme, any desired signal output sequence located on RP is produced. Because the signal output sequence can be designed at will, the fluorescence signal output can be achieved through the CRISPR / Cas12a system with the help of the FQ probe; or by forming a G-quadruplex / hemin complex with hemin, the chemical colorimetric signal output can be achieved with the help of tetramethylbenzidine.

[0076] Example 2

[0077] The specific implementation method of constructing the biosensor in this embodiment refers to Example 1 to verify the feasibility of the biosensor based on the AUW-SDA-CRISRP / Cas12a efficient fluorescence characterization system. The specific steps are as follows:

[0078] 1) Prepare AUW-SDA reaction solution: 1μL 800nM SARS-CoV-2 sequence standard solution, 1μL 1μM SP-9-6, 1μL 1μM RP-CRISPR / Cas-6-2, 1μL 10mM dNTP, 0.4μL 1U / μL Nt.BbvCI nicking endonuclease, 0.5μL 0.5U / μL KF polymerase (3'→5'Exo-) were mixed in CutSmart buffer (50mM potassium acetate, 20mM Tris-Acetate, 10mM magnesium acetate, 100μg / mL BSA, pH 7.9), and the final volume was 20μL.

[0079] 2) The AUW-SDA reaction solution was reacted at 37°C for 3 h, and then incubated at 80°C for 20 min to terminate the reaction.

[0080] 3) The CRISPR / Cas12a reaction solution consisted of 0.25 μL 2300 μg / μL LbCas12a protein and 0.5 μL 200 nM crRNA mixed in NEB 2.1 buffer (10 mM Tris-HCl, 50 mM sodium chloride, 10 mM magnesium chloride, pH 7.9, 100 μg / ml BSA), with a final volume of 15.9 μL, and incubated at 25 °C for 25 min;

[0081] 4) Take 10 μL of CRISPR / Cas12a reaction solution, add 1 μL of 10× Native-PAGE Loading Buffer, use Native-PAGE, electrophoresis at 90 V for 120 min, and analyze the sample;

[0082] 5) Take 4 μL of diluted AUW-SDA reaction solution and add it to the above mixture, and add 0.1 μL of FQ probe, mix well, incubate at 37°C for 30 min, and then incubate at 70°C for 15 min to terminate the reaction. Detect the excitation light spectrum of the CRISPR / Cas12a reaction solution under blue light.

[0083] Comparative Example 1

[0084] The difference between this comparative example and Example 2 is that AUW-SDA reaction solutions with different components are prepared: AUW-SDA reaction solution lacking two enzymes, AUW-SDA reaction solution lacking Nt.BbvCI nicking endonuclease, AUW-SDA reaction solution lacking KF polymerase (3'→5'Exo-), and AUW-SDA reaction solution lacking target, and the other steps are the same. Comparison of the detection effect of the biosensor based on the AUW-SDA-CRISRP / Cas12a efficient fluorescence characterization system, such as Figure 2 shown.

[0085] Depend on Figure 2 It can be seen that only when all components are complete, the excitation light with a peak value of 519nM is highlighted, and the target product can be produced. In the absence of the target or any enzyme, no target product is produced.

[0086] Example 3

[0087] Based on the AUW-SDA-CRISRP / Cas12a efficient fluorescence characterization system, a standard curve of the novel coronavirus nucleic acid sequence and the fluorescence intensity difference (F-F0) was drawn:

[0088] 1) The novel coronavirus nucleic acid sequence SARS-CoV-2 used is shown in SEQ ID No. 24. 1 μL of novel coronavirus nucleic acid sequence standard solution of different concentrations (0, 200 pM, 400 pM, 800 pM, 2 nM, 4 nM, 8 nM, 20 nM, 40 nM, 80 nM) was added to 1 μL 1 μM SP-9-6, 1 μL 1 μM RP-CRISPR / Cas-6-2, 1 μL 10 mM dNTP, 0.4 μL 1 U / μL Nt.BbvCI nicking endonuclease, 0.5 μL 0.5 U / μL KF polymerase (3'→5'Exo-) and mixed in CutSmart buffer (50 mM potassium acetate, 20 mM Tris-Acetate, 10 mM magnesium acetate, 100 μg / mL BSA, pH 7.9), and the final volume was 20 μL.

[0089] 2) The AUW-SDA reaction solution was reacted at 37°C for 3 h, and then incubated at 80°C for 20 min to terminate the reaction.

[0090] 3) The CRISPR / Cas12a reaction solution consisted of 0.25 μL 2300 μg / μL LbCas12a protein and 0.5 μL 200 nM crRNA mixed in NEB 2.1 buffer (10 mM Tris-HCl, 50 mM sodium chloride, 10 mM magnesium chloride, pH 7.9, 100 μg / ml BSA), with a final volume of 15.9 μL, and incubated at 25 °C for 25 min;

[0091] 4) Take 4 μL of the diluted AUW-SDA reaction solution using the designed replacement primer RP and add it to the above mixture, and add 0.1 μL of FQ probe, mix well and incubate at 37°C for 30 min, then incubate at 70°C for 15 min to terminate the reaction, and detect the excitation light intensity of 519 nm of the CRISPR / Cas12a reaction solution under blue light;

[0092] 5) Taking the fluorescence intensity when the concentration of the new coronavirus nucleic acid sequence is 0 pM as F0, calculate the difference in fluorescence intensity at each concentration (ΔF=F-F0), and draw a standard curve of the logarithm of the new coronavirus nucleic acid sequence concentration and the fluorescence intensity difference (ΔF).

[0093] like Figure 3 As shown, the fluorescence intensity difference (ΔF) has a good linear relationship with the logarithm of the concentration of the new coronavirus nucleic acid sequence, the linear range is 5fM to 1pM, and the linear equation is y=41.6321×Ln(Lgx / 0.5970)+22.7259, where x represents the concentration of the new coronavirus nucleic acid sequence, y represents the fluorescence intensity difference (ΔF), and the correlation coefficient R of the linear regression equation is 2 >0.9989, and the limit of detection (LOD) is 0.679fM.

[0094] Example 4

[0095] The specific implementation method of constructing the biosensor in this embodiment refers to Example 1, and the feasibility of the biosensor based on the AUW-SDA-G4 / Hemin high-efficiency chemical color development characterization system is verified. The specific steps are as follows:

[0096] 1) Prepare AUW-SDA reaction solution: the SARS-CoV-2 nucleic acid sequence of the new coronavirus used is shown in SEQ ID No. 24, 1 μL 800nM new coronavirus nucleic acid virus sequence standard solution, 1 μL 1μM SP-9-6, 1μL 1μM RP-CRISPR / Cas-7-2, 1μL 10mM dNTP, 0.4μL 1U / μL Nt.BbvCI nicking endonuclease, 0.5μL 0.5U / μL KF polymerase (3'→5'Exo-) are mixed in CutSmart buffer (50mM potassium acetate, 20mM Tris-Acetate, 10mM magnesium acetate, 100μg / mL BSA, pH 7.9), and the final volume is 20μL.

[0097] 2) The AUW-SDA reaction solution was reacted at 37°C for 3 h, an equal volume of 2×AUW-SDA-G4 buffer (40 mM HEPES, 600 mM ammonium chloride, 0.05% Triton X-100, 2% dimethyl sulfoxide) was added, and incubated at 80°C for 20 min to stop the AUW-SDA reaction, and the temperature was gradually lowered to 25°C.

[0098] 3) Take 20 μL of the reaction solution in 2), add 1 μL of 5 μM Hemin and 1 μL of 2×AUW-SDA-G4 buffer, mix well and incubate at 25°C for 1 h.

[0099] 4) Take 10 μL of the reaction solution, add 1 μL of 10× Native-PAGE Loading Buffer, use Native-PAGE, electrophoresis at 90 V for 120 min, and analyze the sample;

[0100] 5) To the mixture incubated in 3), add 100 μL 2×AUW-SDA-TMB color development buffer (52.4 mM citric acid, 102.8 mM disodium hydrogen phosphate dodecahydrate, 50 mM potassium chloride, 400 mM ammonium chloride), 0.5 μL 200 mM tetramethylbenzidine, dilute to 160 μL with double distilled water, add 40 μL 3% hydrogen peroxide solution and mix well to start the chemical color development reaction, incubate at 25°C for 5 min, add 200 μL 2M hydrochloric acid to terminate the color development reaction, and detect the absorbance spectrum of the reaction solution.

[0101] Comparative Example 2

[0102] The difference between this comparative example and Example 4 is that AUW-SDA reaction solutions with different components are prepared: AUW-SDA reaction solution lacking two enzymes, AUW-SDA reaction solution lacking Nt.BbvCI nicking endonuclease, AUW-SDA reaction solution lacking KF polymerase (3'→5'Exo-), and AUW-SDA reaction solution lacking target, and the other steps are the same. Comparison of the detection effect of the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical color characterization system, such as Figure 4 shown.

[0103] Depend on Figure 4 It can be seen that only when all components are complete, the absorbance with a peak value of 450nM is highlighted, and the target product can be produced. In the absence of the target or any enzyme, no target product is produced.

[0104] Example 5

[0105] Optimization of biosensor conditions based on AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system:

[0106] 1) Take several different reaction conditions and optimize the biosensor conditions by controlling a single variable. The optimization items include: the length of the sequence probe SP loop (such as SP-NX, i.e., SEQ ID No. 1, SEQ ID No. 7-10), the length of the primer region in the replacement primer RP (such as RP-G4 / Hemin-NX, i.e., SEQ ID No. 4, SEQ ID No. 11-14), the length of the foot in the replacement primer RP (such as RP-G4 / Hemin-XN, i.e., SEQ ID No. 4, SEQ ID No. 15-18), the concentration of the replacement primer RP, the amount of Nt.BbvCI nicking endonuclease added, the amount of KF polymerase added, the reaction time of SDA-AUW, the concentration of hemin in the color reaction, and the color development time in the color reaction. The sequences involved are shown in Table 1.

[0107] 2) The standard reaction system is: different concentrations of new coronavirus nucleic acid virus sequence standard solution, the new coronavirus nucleic acid sequence SARS-CoV-2 used is shown in SEQ ID No. 24, 1μL 1μM SP, 1μL 1μM RP, 1μL 25mM dNTP, 0.4μL 1U / μL Nt.BbvCI nicking endonuclease, 0.5μL 0.5U / μL KF polymerase (3'→5'Exo-) are mixed in CutSmart buffer (50mM potassium acetate, 20mMTris-Acetate, 10mM magnesium acetate, 100μg / mL BSA, pH7.9), and the final volume is 20μL.

[0108] 3) Take 20 μL of the reaction solution in 2), add 1 μL of 5 μM hemin and 1 μL of 2×AUW-SDA-G4 buffer, mix well and incubate at 25°C for 1 h.

[0109] 4) To the mixture incubated in 3), add 100 μL 2×AUW-SDA-tetramethylbenzidine colorimetric buffer (52.4 mM citric acid, 102.8 mM disodium hydrogen phosphate dodecahydrate, 50 mM potassium chloride, 400 mM ammonium chloride), 0.5 μL 200 mM tetramethylbenzidine, dilute to 160 μL with double distilled water, add 40 μL 3% hydrogen peroxide solution and mix well to start the chemical colorimetric reaction, incubate at 25° C. for 5 min, add 200 μL 2 M hydrochloric acid to terminate the colorimetric reaction; detect the absorbance of the reaction solution at 450 nm.

[0110] The optimization results are as follows Figure 5The optimal reaction conditions were determined: the loop length of the sequence probe SP was 9, the primer region length of the displacement primer RP was 7, the foot length of the displacement primer RP was 2, the concentration of the displacement primer RP was 50 nM, the addition amount of Nt.BbvCI nicking endonuclease was 0.4 U, the addition amount of KF polymerase was 0.2 U, the reaction time of AUW-SDA was 3 hours, the concentration of hemin in the color reaction was 12.5 nM, and the color development time in the color reaction was 5 minutes.

[0111] Example 6

[0112] Based on the AUW-SDA-G4 / Hemin pathway, a standard curve of the difference between the nucleic acid sequence of the new coronavirus and the absorbance (A-A0) was drawn:

[0113] 1) Take several different concentrations of new coronavirus nucleic acid virus sequence standard solutions, the new coronavirus nucleic acid sequence SARS-CoV-2 used is shown in SEQ ID No. 24, and make the reaction solution have a gradient final concentration (0, 200pM, 400pM, 800pM, 2nM, 4nM, 8nM, 20nM, 40nM, 80nM, 200nM), add 1μL 1μM SP-9-6, 1μL 1μM RP-G4 / hematin chloride-7-2, 1μL 10mM dNTP, 0.4μL 1U / μL Nt.BbvCI nicking endonuclease, 0.4μL 0.5U / μL KF polymerase (3'→5'Exo-) and mix in CutSmart buffer (50mM potassium acetate, 20mM Tris-Acetate, 10mM magnesium acetate, 100μg / mL BSA, pH 7.9), the final volume was 20 μL.

[0114] 2) The AUW-SDA reaction solution was reacted at 37°C for 3 h, an equal volume of 2×AUW-SDA-G4 buffer (40 mM HEPES, 600 mM ammonium chloride, 0.05% Triton X-100, 2% dimethyl sulfoxide) was added, and incubated at 80°C for 20 min to stop the AUW-SDA reaction, and the temperature was gradually lowered to 25°C.

[0115] 3) Take 20 μL of the reaction solution in 2), add 1 μL of 5 μM Hemin and 1 μL of 2×AUW-SDA-G4 buffer, mix well and incubate at 25°C for 1 h.

[0116] 4) Add 100 μL 2×AUW-SDA-TMB color development buffer (52.4 mM citric acid, 102.8 mM sodium phosphate dibasic dodecahydrate, 50 mM potassium chloride, 400 mM ammonium chloride) and 0.5 μL 200 mM tetramethylbenzidine to the mixture after incubation in 3), dilute to 160 μL with double distilled water, add 40 μL 3% hydrogen peroxide solution and mix well to start the chemical color development reaction, incubate at 25°C for 5 min, and add 200 μL 2M hydrochloric acid to terminate the color development reaction. Detect the absorbance of the reaction solution at 450 nm.

[0117] 5) Taking the absorbance at 450nm when the concentration of the new coronavirus nucleic acid sequence is 0pM as A0, calculate the absorbance difference at each concentration (ΔA=A-A0), and draw a standard curve of the logarithm of the new coronavirus nucleic acid sequence concentration and the absorbance difference (ΔA).

[0118] like Figure 6 As shown, the absorbance difference (ΔA) has a good linear relationship with the logarithm of the concentration of the new coronavirus nucleic acid sequence, the linear range is 250fM to 10pM, and the linear equation is y=0.00173×Ln(Lgx / 0.776)+0.00597, where x represents the concentration of the new coronavirus nucleic acid sequence, y represents the absorbance difference (ΔA), and the correlation coefficient R of the linear regression equation is 2 >0.997, with a limit of detection (LOD) of 13.5 fM.

[0119] Example 7

[0120] Application testing:

[0121] The reliability test of the novel coronavirus nucleic acid was performed on the dual-signal biosensor based on AUW-SDA designed in Examples 2 and 4 of the present invention. The test was performed by adding the novel coronavirus N gene nucleic acid standard to the pharyngeal swab. The novel coronavirus N gene standard used was obtained by T7 in vitro transcription (for specific methods, see ACS Sensor, 2021, 6, 1086-1093). The pharyngeal swab was sampled from healthy volunteers, and the pharyngeal swab was immersed in the viral nucleic acid preservation solution (RNase-free PBS buffer, RNase inhibitor 0.1U / μL, pH 7.4). The virus preservation solution after immersion in the pharyngeal swab was spiked with the novel coronavirus N gene nucleic acid standard (500pM, 200pM, 50pM, 10pM, 4pM, 0.6pM), and the standard curve was established using the present invention, and the spiked samples were detected.

[0122] The biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system detects target concentrations of 10pM, 4pM, and 0.6pM, and optimizes the length of the sequence probe SP foot (e.g., SP-XN, i.e., SEQ ID No. 1-2, SEQ ID No. 19-23) to improve the ability to detect long sequences, such as Figure 7 As shown in A, the optimal length of the sequence probe SP foot is 14; establish a standard curve, such as Figure 7 As shown in B, the linear range is 20fM to 12pM, and the linear equation is y=14.4×Ln(Lgx / 0.798)+55.3, where x represents the concentration of the new coronavirus nucleic acid sequence, and y represents the fluorescence intensity difference (ΔF). The correlation coefficient R of the linear regression equation 2 >0.991.

[0123] The target concentrations of the biosensor based on the AUW-SDA-G4 / Hemin high-efficiency chemical colorimetric characterization system are 500pM, 200pM, and 50pM. The standard curve of the detection results is shown in Figure 8 As shown, the length of the sequence probe SP foot (eg, SP-XN, ie, SEQ ID No. 1-2, SEQ ID No. 19-23) is optimized to improve the ability to detect long sequences, as shown in Figure 8 As shown in A, the optimal length of the sequence probe SP foot is 14; establish a standard curve, such as Figure 8 As shown in Figure B, the linear range is 1pM to 3nM, and the linear equation is y=0.00186×Ln(Lgx)+0.0201, where x represents the concentration of the new coronavirus nucleic acid sequence, y represents the absorbance difference (ΔA), and the correlation coefficient R of the linear regression equation is 2 >0.997.

[0124] Comparative Example 3

[0125] The difference between this comparative example and Example 7 is that RT-qPCR is used as a control for the two biosensors. The spike recovery results of the biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system, the biosensor based on the AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system in Example 7 and the RT-qPCR in this comparative example are shown in Table 2. Figure 7 C, as shown in 8C.

[0126] Table 2 Comparison of spike recovery results

[0127]

[0128] Depend on Figure 7 C and Figure 8C shows that there is no significant difference between the two methods and the RT-qPCR detection results. This shows that the biosensors based on AUW-SDA-CRISRP / Cas12a and AUW-SDA-G4 / Hemin have a detection level comparable to that of RT-qPCR, and are simple to operate without relying on expensive instruments, especially the biosensor based on AUW-SDA-G4 / Hemin, which can read the detection results directly with the naked eye.

[0129] Comparative Example 4

[0130] Specific detection of biosensors based on the AUW-SDA-CRISRP / Cas12a efficient fluorescence characterization system

[0131] The biosensor based on the AUW-SDA-CRISPR / Cas12a efficient fluorescence characterization system of Example 2 of the present invention was used to detect a series of virus sequences, including SARS as shown in SEQ ID No.25, MERS as shown in SEQ ID No.26, H1N1 as shown in SEQ ID No.27, H7N9 as shown in SEQ ID No.28, the concentration of the new coronavirus sequence was set to 20pM, the new coronavirus nucleic acid sequence SARS-CoV-2 used was shown in SEQ ID No.24, and the concentration of other viral nucleic acid sequences was: 200pM. Characterization was performed using CRISPR / Cas12a fluorescence colorimetry.

[0132] Specific test results such as Fig. 9 As shown. Fig. 9 It can be seen that the biosensor based on AUW-SDA-CRISRP / Cas12a has a good response to the nucleic acid sequence of the new coronavirus, and has no obvious response to other viral nucleic acid sequences at ten times the concentration. The biosensor based on the AUW-SDA-CRISRP / Cas12a efficient fluorescence characterization system is specific to the nucleic acid sequence of the new coronavirus, and other viral nucleic acid sequences will not affect the detection results of the nucleic acid sequence of the new coronavirus.

[0133] Comparative Example 5

[0134] Specific detection of biosensors based on AUW-SDA-G4 / Hemin efficient chemical colorimetric characterization system

[0135] The biosensor based on the AUW-SDA-G4 / Hemin efficient chemical color characterization system of Example 4 of the present invention was used to detect a series of virus sequences, including SARS as shown in SEQ ID No.25, MERS as shown in SEQ ID No.26, H1N1 as shown in SEQ ID No.27, and H7N9 as shown in SEQ ID No.28. The concentration of the new coronavirus sequence was set to 1 nM, the new coronavirus nucleic acid sequence SARS-CoV-2 used was shown in SEQ ID No.24, and the concentration of other viral nucleic acid sequences was 10 nM. AUW-SDA-G4 / Hemin chemical color development was used for characterization.

[0136] Specific test results such as Fig.10 As shown. Fig.10 It can be seen that the biosensor based on AUW-SDA-G4 / Hemin has a good response to the nucleic acid sequence of the new coronavirus, and has no obvious response to other viral nucleic acid sequences at ten times the concentration. The biosensor based on the AUW-SDA-G4 / Hemin high-efficiency chemical colorimetric characterization system is specific to the nucleic acid sequence of the new coronavirus, and other viral nucleic acid sequences will not affect the detection results of the nucleic acid sequence detection biosensor of the new coronavirus described in the present invention.

[0137] In summary, the AUW-SDA technology in the present invention can uniformly realize activation of amplification without relying on the 3' end of the target, and can be used to identify long sequences; realize target replacement to obtain higher sensitivity and reaction efficiency; by designing variable sequences on the RP sequence, any nucleic acid signal output sequence can be amplified to adapt to a variety of nucleic acid-based biosensors. This technology has been adapted to CRISPR / Cas12a nucleic acid biosensors and G4 / Hemin-TMB nucleic acid biosensors. The nucleic acid signal output based on AUW-SDA has an extremely high linear range and can be used for more universal detection, which solves the main shortcomings of traditional SDA technology used in biosensors, and realizes efficient, universal and convenient adaptation of a variety of nucleic acid biosensors to output signals. Different nucleic acid targets can be detected by simply replacing the sequence probe SP and the replacement primer RP; and different nucleic acid biosensors can be adapted by only changing the replacement primer RP.

[0138] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A dual-signal biosensor based on AUW-SDA technology, characterized in that: include, A sequence probe, wherein the sequence probe comprises a first hybridization region and a second hybridization region, wherein the first hybridization region and the second hybridization region are connected by a loop region to form a hairpin structure; the first hybridization region contains a sequence complementary to a target, and the target binds to the sequence of the first hybridization region to open the hairpin structure; the sequence of the loop region contains a nicking endonuclease site; A displacement primer, wherein the displacement primer contains a sequence complementary to the second hybridization region and a sequence complementary to the product strand, and the displacement primer binds to the sequence probe to form a first 3' end that can be recognized and paired by a KF polymerase lacking 3'→5' exonuclease activity; A nicking endonuclease for cleaving the sequence probe to form a second 3' end that can be recognized and paired by a KF polymerase lacking 3'→5' exonuclease activity; The KF polymerase lacking 3'→5' exonuclease activity pairs with the first 3' end to initiate polymerization, and uses the sequence probe as a template to synthesize a corresponding complementary sequence to synthesize a sequence probe-displacement primer complex; the KF polymerase lacking 3'→5' exonuclease activity pairs with the second 3' end to initiate polymerization, and uses the displacement primer as a template to synthesize a corresponding product sequence; Wherein, the nicking endonuclease cuts the sequence probe to generate a product sequence.

2. A method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 1, characterized in that: include, When the sequence probe does not recognize the target, the hairpin structure is maintained, and the displacement primer has difficulty in invading the sequence probe in the hairpin configuration to form the first 3' end of the pair, and it cannot be recognized by the KF polymerase lacking 3'→5' nuclease activity; When the sequence probe recognizes the target, the target binds to the sequence probe, the sequence probe opens the hairpin structure, and the displacement primer binds to the sequence probe to form a paired first 3' end that can be recognized by the KF polymerase lacking 3'→5' exonuclease activity; KF polymerase lacking 3'→5' exonuclease activity initiates polymerization to complete the complementary sequence at the nicking enzyme site on the sequence probe. At the same time, the corresponding complementary sequence is synthesized using the sequence probe as a template, displacing the target already bound to the sequence probe, and synthesizing a sequence probe-displacement primer complex with a complete nicking enzyme site. The displaced target enters the next cycle; The sequence probe-displacement primer complex makes an incision at the incision endonuclease site under the action of the incision endonuclease, forming a second 3' end that can be recognized by the KF polymerase lacking 3'→5' nuclease activity; Under the action of KF polymerase lacking 3'→5' exonuclease activity, a strand displacement amplification reaction occurs at the incision, and the corresponding product sequence is synthesized using the displacement primer as a template; The product sequence depends on the sequence on the displacement primer that is complementary to the product chain and is completely independent of the target sequence. By designing the sequence on the displacement primer that is complementary to the product chain, any product sequence can be amplified to adapt to a variety of nucleic acid-based biosensors to detect target concentrations.

3. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 2, characterized in that: The nucleic acid sequence corresponding to the sequence probe is shown in one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.7-10, and SEQ ID No.19-23.

4. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 2 or 3, characterized in that: The sequence on the replacement primer that is complementary to the product chain is the complementary sequence of the CRISPR / Cas12a system activator, thereby realizing the output of the fluorescent signal.

5. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 4, characterized in that: The nucleic acid sequence of the replacement primer is shown in SEQ ID No.

3.

6. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 5, characterized in that: When used for the detection of novel coronavirus nucleic acid, the novel coronavirus nucleic acid is used as the target. The novel coronavirus nucleic acid sequence standard solutions of different concentrations are mixed with the AUW-SDA reaction solution in CutSmart buffer for reaction. Then, the CRISPR / Cas12a reaction solution is added, and the FQ probe is added. The reaction is mixed, and the excitation light intensity is detected to draw a standard curve and determine the concentration. Wherein, the nucleic acid sequence corresponding to the FQ probe is shown as SEQ ID No.

5.

7. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 6, characterized in that: The components of the AUW-SDA reaction solution are 10-100 nM sequence probe, 10-100 nM replacement primer, 0.5-2.5 mM dNTP, 0.005-0.1 U / μL nicking endonuclease and 0.005-0.02 U / μL KF polymerase lacking 3'→5' exonuclease activity, and the solution system is CutSmart buffer; The CutSmart buffer composition is 50 mM potassium acetate, 20 mM Tris-Acetate, 10 mM magnesium acetate and 100 μg / mL BSA, pH 7.9; the AUW-SDA reaction solution is reacted at 37° C. for 1 to 4 hours, and the reaction is terminated by incubation at 70 to 95° C. for 5 to 25 minutes.

8. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 2 or 3, characterized in that: The sequence on the displacement primer that is complementary to the product chain is the complementary sequence of the G-quadruplex, thereby realizing the output of chemical color development signals.

9. The method for detecting target concentration using a dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 8, characterized in that: The nucleic acid sequence of the replacement primer is shown in one of SEQ ID No. 4 and SEQ ID No. 11-18.

10. The method for detecting target concentration using the dual-signal biosensor constructed based on AUW-SDA technology as claimed in claim 9, characterized in that: When used for the detection of novel coronavirus nucleic acid, novel coronavirus nucleic acid sequence standard solutions of different concentrations are mixed with AUW-SDA reaction solution in CutSmart buffer for reaction, and then Hemin is added and mixed in AUW-SDA-G4 buffer; then TMB and hydrogen peroxide solution are added and mixed in AUW-SDA-TMB color development buffer for color development reaction, and the absorbance of the reaction solution is detected to draw a standard curve and determine the concentration; The components of the AUW-SDA-G4 buffer are 10-20 mM HEPES, 50-400 mM ammonium chloride, 0.0125-0.05% Triton X-100 and 0.5-1% dimethyl sulfoxide; the components of the AUW-SDA-TMB color development buffer are 26.2 mM citric acid, 51.4 mM disodium hydrogen phosphate dodecahydrate, 10-50 mM potassium chloride and 50-400 mM ammonium chloride.

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