A coded test strip and kit based on spherical nucleic acid detection probes and their applications

Abstract

The present invention discloses a coded test strip and kit based on a spherical nucleic acid detection probe and its application. The spherical nucleic acid detection probe is based on gold nanoparticles, and polyA-DNA is fixed on the surface of the gold nanoparticles by adsorption of polyA bases. The polyA-DNA consists of single-stranded DNA and a polyA sequence at its 3' end. The sequence of the single-stranded DNA is shown in SEQ ID NO: 1, and the number of polyA bases is 2-20. Through the adsorption of A bases by gold nanoparticles, the present invention can connect DNA to the surface of gold nanoparticles without additional modification, thereby simplifying the preparation process of the spherical nucleic acid detection probe and reducing the detection cost. At the same time, by changing the polyA length, the density and spacing of the spherical nucleic acid surface probes are regulated, thereby improving the detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a coded test strip and a kit based on spherical nucleic acid detection probes and their applications. Background Art

[0002] Rapid and accurate disease diagnosis methods can save time, money, and labor costs, improve medical efficiency, and reduce misdiagnosis and treatment costs. Currently, the most commonly used screening test for diseases caused by infectious pathogens is antigen test strips. Although antigen test strips offer the advantages of portability and visual detection, they have low sensitivity and poor accuracy, making it difficult to accurately detect infectious diseases.

[0003] Combining nucleic acid amplification technology with nucleic acid detection test strips can significantly improve the sensitivity of pathogen detection. Currently used nucleic acid detection test strips are mainly divided into two types. The first type is nucleic acid detection test strips based on antigen-antibody specific binding, and the second type is nucleic acid detection test strips based on base complementary pairing. Nucleic acid detection test strips based on antigen-antibody specific binding may produce false positive signals due to primer dimers or non-specific amplification during the actual detection process, resulting in low accuracy. Non-antibody nucleic acid test strips that recognize amplification products based on base complementary pairing have good specificity and have great advantages in detecting single-base mutations. However, the current preparation methods of spherical nucleic acid probes used in such test strips, such as salt aging and freezing methods, require thiol modification of the nucleotide chains used, which is time-consuming and costly. This spherical nucleic acid constructed by chemical coupling has densely distributed nucleic acids on the surface, and the electrostatic repulsion and strong steric hindrance between adjacent oligonucleotides cause a large number of recognition sites to be unable to function, which may increase the difficulty of recognition and thus reduce the detection sensitivity. In addition, when used for nucleic acid testing, conventional nucleic acid amplification products are difficult to be directly combined with test strips and need to be denatured at high temperature or digested with uracil DNA glycosylase to generate a single-stranded structure before testing.

[0004] Therefore, the development of a low-cost, sensitive and accurate nucleic acid-encoded test strip detection method is of great significance for the highly sensitive and immediate detection of pathogens. Summary of the Invention

[0005] The present invention aims to solve one of the related technical problems to at least a certain extent. To this end, the embodiments of the present invention provide a coded test strip and a kit based on spherical nucleic acid detection probes and their applications.

[0006] The technical solutions adopted in the embodiments of the present invention are as follows:

[0007] Firstly,

[0008] An embodiment of the present invention provides a coded test strip based on a spherical nucleic acid detection probe (for instant nucleic acid detection). The spherical nucleic acid detection probe has a gold nanoparticle as its core, and polyA-DNA is immobilized on the surface of the gold nanoparticle by adsorption of poly A bases. The polyA-DNA consists of a single-stranded DNA and a poly A sequence at its 3' end. The sequence of the single-stranded DNA is shown in SEQ ID NO: 1, and the number of poly A bases is 2-20.

[0009] 5'-TTTTTTTTTCACCATGACAGTGTAAGATCGACATAGG-3'(SEQ ID NO:1)

[0010] The spherical nucleic acid detection probes on the coded test strips of the present invention are centered around gold nanoparticles, with polyA-DNA oligonucleotide chains adsorbed onto the gold nanoparticle surfaces via polyA bases. By regulating the length of the polyA, the present invention optimizes the density of oligonucleotide chains adsorbed onto the gold nanoparticle surfaces, systematically adjusting the surface probe density and lateral spacing on the gold nanoparticles, effectively improving the binding efficiency between the spherical nucleic acid detection probes and the analyte. The present invention also adds a poly T sequence to the 5' end of the polyA-DNA, leveraging its relatively weak binding to gold nanoparticles to maintain the adsorbed polyA-DNA in a relatively upright position, promoting its binding to the T-line and C-line probes. The central sequence of the probe is a coding region designed to specifically recognize and bind to the terminal sequence of the ISP18-modified hairpin primer.

[0011] Preferably, the number of bases of the poly A is 7-15, more preferably, the number of bases of the poly A is 10; in this case, the sensitivity of the test strip is significantly improved.

[0012] The coded test strip of the present invention is a universal nucleic acid detection test strip, comprising a base plate, and a sample pad, a gold label pad, a nitrocellulose membrane, and a water-absorbing pad, which are sequentially overlapped on the base plate. The gold label pad is coated with the spherical nucleic acid detection probe, and the nitrocellulose membrane is provided with a test line (T line) and a quality control line (C line).

[0013] In some embodiments, the sample pad is stacked 1-2 mm above the gold label pad, the gold label pad is stacked 1-2 mm above the nitrocellulose membrane, and the absorbent pad is stacked 1-2 mm above the nitrocellulose membrane.

[0014] In some embodiments, the detection line is coated with streptavidin and 3' end biotinylated DNA T The quality control line is coated with streptavidin and 3' end biotinylated DNA C .

[0015] In some embodiments, DNA T The sequence is shown in SEQ ID NO: 2-Biotin, the DNA C The sequence is shown in SEQ ID NO: 3-Biotin.

[0016] 5'-CGCTGATAGATAGGCTTGTG-3' (SEQ ID NO: 2),

[0017] 5'-TGGTGAAAAAAAAAA-3'(SEQ ID NO:3)

[0018] The detection line on the coded test strip of the present invention is coated with streptavidin and biotinylated DNA T , the quality control line is coated with streptavidin and biotinylated DNA C , the DNA T is a coding sequence, the DNA C The 5' end of the test strip is a coding sequence, and the 3' end is a polyA sequence. The introduction of the coding sequence reduces the non-specific binding that may occur when the test strip is used, thereby improving the detection accuracy.

[0019] Secondly,

[0020] The present invention also provides a method for preparing the above-mentioned coded test strip based on the spherical nucleic acid detection probe, comprising the following steps:

[0021] S1. Preparation of spherical nucleic acid detection probes;

[0022] The gold nanoparticle (AuNPs) solution and the polyA-DNA solution were mixed in a glass bottle, and the glass bottle was placed in a microwave oven and heated for 5 minutes, and then ultrapure water was added to dissolve it, and after centrifugation, washing was repeated three times, and finally the precipitate was resuspended in 1×PBS to obtain the spherical nucleic acid detection probe;

[0023] S2, bind streptavidin to 3' end biotinylated DNA T After incubation, the T-line probe was obtained and streptavidin was bound to the 3'-end biotinylated DNA. C After mixed incubation, the C-line probe was obtained;

[0024] S3. Absorb the spherical nucleic acid detection probe and evenly apply it to the gold label pad; dip the T-line probe and C-line probe respectively, and draw T lines and C lines respectively on the nitrocellulose membrane; overlap the sample pad, gold label pad, nitrocellulose membrane, and absorbent pad on the bottom plate in sequence, and cut them to obtain the coded test strip.

[0025] In some embodiments, in step S1, the centrifugation is performed at 4°C, 10,000 rpm for 10 minutes, and the washing is performed with 1×PBS.

[0026] In some embodiments, in step S1, the gold nanoparticles are prepared using a chloroauric acid-sodium citrate reduction method. The gold nanoparticle solution is prepared by magnetically stirring 0.005%-0.05% (v / v) chloroauric acid in an oil bath, heating under reflux until boiling, rapidly adding 0.5%-5% (w / w) sodium citrate, continuing heating and stirring for 15 minutes, and then cooling to room temperature, wherein the volume ratio of chloroauric acid to sodium citrate is 25:1.

[0027] In some embodiments, in step S1, the concentration of polyA-DNA in the polyA-DNA solution is 100 μM.

[0028] In some embodiments, in step S1, the volume ratio of the gold nanoparticle solution to the polyA-DNA solution is (10-20):1.

[0029] In some embodiments, in step S2, the concentration of streptavidin is 1 mg / mL; the biotinylated DNA T The concentration was 100 μM, and the biotinylated DNA C The concentration is 100 μM.

[0030] In some embodiments, in step S2, the streptavidin binds to the biotinylated DNA T and DNA C The volume ratio is (0.1-2):1.

[0031] In some embodiments, in step S2, the incubation condition is incubating at 4°C for 1 hour.

[0032] In some embodiments, the dosage of the spherical nucleic acid detection probe is 2-12 μL / cm, and the dosage of the T-line probe and the C-line probe are both 0.1-1 μL / cm.

[0033] Thirdly,

[0034] An embodiment of the present invention also provides a detection card, which includes the above-mentioned test strip and a card shell arranged on the outside of the test strip, the card shell includes an upper card shell and a lower card shell that are locked with each other, the lower card shell is provided with a card slot for placing the test strip, the upper card shell is provided with a sample addition port corresponding to the sample pad of the test strip, and is provided with an observation window corresponding to the nitrocellulose membrane of the test strip, and the detection line and quality control line on the nitrocellulose membrane are both exposed at the observation window.

[0035] Fourthly,

[0036] The present invention also provides a kit for detecting a novel coronavirus, comprising:

[0037] The above-mentioned test strips or test cards;

[0038] A forward primer, which is modified with ISP18 and has a hairpin structure at the 5' end; the forward primer is: 5'-SEQ ID NO:4 / ISP18 / SEQ ID NO:5-3';

[0039] 5'-AAGCCTCTTCTCGT-3'(SEQ ID NO:4)

[0040] 5'-ACGAGAAGAGGCTTGTGGTGACGGTAAAATGAAAGATCTCAGTC-3'(SEQ ID NO:5)

[0041] A reverse primer, which is modified with ISP18 and has a hairpin structure at the 5' end; the reverse primer is: 5'-SEQ ID NO:6 / ISP18 / SEQ ID NO:7-3';

[0042] 5'-TCACGTAGTCGCAA-3'(SEQ ID NO:6)

[0043] 5'-TTGCGACTACGTGACTGCCTGGAGTTGAATTTCTTGAACTGTTG-3' (SEQ ID NO: 7).

[0044] The PCR primers in the kit of the present invention are modified with ISP18, and the amplified products can be directly used for nucleic acid test strip detection. The nucleic acid detection method using the kit of the present invention has the advantages of simple operation, low cost, high sensitivity and good specificity, and can be used for real-time nucleic acid detection with good practicality.

[0045] In some embodiments, the kit further comprises:

[0046] Lysis buffer, which is a mixture of 100 mM TCEP and 2 mM EDTA in a volume ratio of 1:1;

[0047] 20 U / μL RNase inhibitor, 2.5 mM dNTP, 40 U / μL reverse transcriptase, 5× reverse transcriptase buffer, 2×PCR Mix.

[0048] Fifthly,

[0049] The embodiments of the present invention also provide the use of the above-mentioned coded test strips, test cards, and kits in nucleic acid detection.

[0050] Sixth aspect,

[0051] The present invention also provides a nucleic acid detection method based on a spherical nucleic acid detection probe coded test strip, comprising the following steps: extracting nucleic acid from a test sample; amplifying a target fragment in the test sample by a PCR reaction; and dripping the amplified product onto the test strip to obtain a detection result.

[0052] The present invention also provides a nucleic acid detection method based on a spherical nucleic acid detection probe coded test strip, comprising the following steps:

[0053] (1) The sample to be tested was mixed with lysis buffer (100 mM TCEP, 2 mM EDTA) in a 1:1 ratio and lysed at room temperature for 5-10 min. The lysate was used for subsequent experiments.

[0054] (2) The above lysate was reverse transcribed. The reverse transcription reaction system was as follows: 2 μL lysate, 0.5 μL RNase inhibitor (20 U / μL), 2 μL dNTP (2.5 mM), 1 μL reverse transcriptase (40 U / μL), 1 μL reverse primer (10 μM), 2 μL reverse transcriptase buffer (5×), DEPC water was added to 10 μL, and the mixture was incubated at 42°C for 1 h to obtain cDNA.

[0055] (3) The cDNA was subjected to PCR reaction. The PCR reaction system was as follows: 10 μL PCR Mix (2×), 2 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and ddH2O was added to make up to 20 μL. The reaction procedure was denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute, and 40 cycles.

[0056] (4) Add the PCR product to the test strip and read the test results.

[0057] When both the C line and the T line of the test strip are colored, it is positive; when only the C line is colored, it is negative; when the C line does not show color, the test strip is invalid.

[0058] Compared with the closest prior art, the present invention has the following beneficial effects:

[0059] The present invention utilizes the adsorption of poly A bases by gold nanoparticles to connect DNA to the surface of gold nanoparticles without additional modification, thereby simplifying the preparation process of spherical nucleic acid detection probes and reducing detection costs. At the same time, by changing the poly A length, the density and spacing of probes on the spherical nucleic acid surface are regulated, thereby improving detection sensitivity.

[0060] The present invention performs ISP18 modification on primers, and the PCR products can be directly applied to coded test strips based on spherical nucleic acid detection probes, with high accuracy and good specificity in detection results. The method is versatile and can be used for the detection of other viruses and pathogens by simply adjusting the primer sequences accordingly. The test results are output using test strips, achieving visual detection and being user-friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0062] Figure 1 The following are comparison diagrams of the design principles and test results of coded test strips, wherein (a) is a schematic diagram of the principle of preparing spherical nucleic acid detection probes by salt aging and microwave heating methods and using them to construct coded test strips; (b) is the sensitivity of the coded test strip based on poly A-DNA spherical nucleic acid detection probes prepared by microwave heating; (c) is the sensitivity of the coded test strip based on spherical nucleic acid detection probes prepared by salt aging method;

[0063] Figure 2 Figure 3. Characterization of spherical nucleic acid detection probes connected to polyA-DNA of different lengths and the corresponding nucleic acid test strip detection sensitivity. (a) is a schematic diagram of gold nanoparticles connected to polyA-DNA of different lengths, (b) is a diagram of the zeta potential of the spherical nucleic acid detection probes, (c) is a diagram of the hydrated ion radius of the spherical nucleic acid detection probes, and (d) is a comparison of the surface probe density and detection sensitivity.

[0064] Figure 3 Characterization of the hybridization rate and surface DNA spacing of spherical nucleic acid detection probes connected to polyA-DNA of different lengths, where (a) shows the changes in the UV absorption spectrum of spherical nucleic acid detection probes connected to polyA-DNA of different lengths and linker-DNA at 525nm; (b) shows the changes in the fluorescence signal of the spherical nucleic acid detection probes connected to polyA-DNA of different lengths hybridized with the fluorescent probe; (c) shows the calculated spacing of probes on the surface of gold nanoparticles connected to polyA-DNA of different lengths, with the right figure showing a schematic diagram of the spacing; (d) shows the principle diagram of the connection density of polyA-DNA of different lengths on gold nanoparticles;

[0065] Figure 4Figure 2 shows the detection principle and sensitivity and specificity of the spherical nucleic acid detection probe kit. (a) Schematic diagram of the detection principle of PCR combined with a coded test strip; (b) Detection results of the coded test strip using different concentrations of SARS-CoV-2 pseudovirus; (c) ImageJ standard curve for grayscale value determination of the coded test strip; (d) Schematic diagram of the SARS-CoV-2 virus detection process; (e) Detection results of the test strip using an actual sample; (f) PCR detection results of an actual sample. DETAILED DESCRIPTION

[0066] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0067] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0068] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the relevant fields or are performed according to the conditions recommended by the manufacturer.

[0069] The variants of the novel coronavirus SARS-CoV-2 known to date include but are not limited to Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, and Omicron.

[0070] Where values ​​are described as a range, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, regardless of whether a specific value or sub-range is expressly stated.

[0071] As used herein, the term "about" means + / - 10% of the recited value.

[0072] Example 1: Microwave Heating Synthesis of Spherical Nucleic Acid Detection Probes for Construction of Nucleic Acid Test Strips

[0073] (1) Preparation of AuNPs;

[0074] Place 25 mL of 0.01% (v / v) HAuCl4 in an oil bath with magnetic stirring, heat under reflux until boiling, quickly add 1 mL of 1% (w / w) sodium citrate, continue heating and stirring for 15 minutes, then cool to room temperature, store in a glass bottle, wrap in tin foil, and store at 4°C in the dark.

[0075] (2) Preparation of spherical nucleic acid detection probes;

[0076] Spherical nucleic acid detection probes were prepared using microwave heating. 5 μL (100 μM) polyA-DNA solution and 100 μL gold nanoparticle solution were added to a 1.5 mL glass vial and mixed thoroughly. The vial was then microwaved on high for 5 minutes and resuspended in ultrapure water. The solution was washed with 1× PBS and centrifuged at 10,000 rpm for 10 minutes at 4°C. The particles were resuspended in 1× PBS and washed three times. Finally, the particles were wrapped in tin foil and stored at 4°C in the dark.

[0077] PolyA-DNA consists of a single-stranded DNA and a poly A sequence at its 3' end. The sequence of the single-stranded DNA is shown in SEQ ID NO: 1, and the number of bases in the poly A sequence is 2-25.

[0078] In Example 1 of the present invention, spherical nucleic acid detection probes containing different A base lengths were prepared.

[0079] When n=2, the probe is named A2-SNAs, and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO:8:

[0080] 5'-TTTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAA-3' (SEQ ID NO: 8) When n=5, the probe is named A5-SNAs, and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO: 9:

[0081] 5'-TTTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAAAAA-3' (SEQ ID NO: 9) When n=10, the probe is named A10-SNAs, and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO: 10:

[0082] 5'-TTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAAAAAAAAAA-3'(SEQ ID NO:10)

[0083] When n=15, the probe is named A15-SNAs, and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO: 11:

[0084] 5'-TTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAAAAAAAAAAAAAAA-3'(SEQ IDNO:11)

[0085] When n=20, the probe is named A20-SNAs, and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO: 12:

[0086] 5'-TTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAAAAAAAAAAAAAAAA AAAA-3'(SEQID NO:12)

[0087] When n=25, the probe is named A25-SNAs (for comparison), and the nucleotide sequence of polyA-DNA is shown in SEQ ID NO: 13:

[0088] 5'-TTTTTTTTTCACCATGACAGTGTAAGATCGACATAGGAAAAAAAAAAAAAAAA AAAAAAAAA-3'(SEQ ID NO:13)

[0089] Spherical nucleic acid detection probes, named SH-SNAs (for comparison), were prepared using a salt aging method. 100 μL of gold nanoparticle solution, 100 μM SH-DNA (SEQ ID NO: 1 with 3'-end SH modification), and 5 μL of 10% (v / v) SDS were added to 89 μL of ultrapure water, mixed well, and incubated at 37°C in a water bath for 12 hours. 5 M NaCl was then added in batches (once every 2 hours, for a total of 12 hours). Spherical nucleic acid detection probes were obtained after a further 12-hour incubation. The probes were wrapped in tin foil and stored at 4°C in the dark.

[0090] (3) Preparation of T-line probe and C-line probe:

[0091] Streptavidin and biotinylated DNA T and biotinylated DNA C After mixing, incubate at 4°C and store the prepared T-line probe and C-line probe at 4°C. The T-line probe includes streptavidin and 3' end biotinylated DNA. T The C-line probe consists of streptavidin and 3'-end biotinylated DNA C ; Separately bind streptavidin to biotinylated DNA T and biotinylated DNA C After mixing, the mixture was incubated at 4°C, centrifuged at 10,000 rpm for 10 minutes, and the precipitate was washed with 1×PBS, which was repeated three times. Finally, the precipitate was resuspended in 1×PBS to obtain the T-line probe and C-line probe.

[0092] DNA T The sequence is shown in SEQ ID NO: 2-Biotin, the DNAC The sequence is shown in SEQ ID NO: 3-Biotin.

[0093] 5'-CGCTGATAGATAGGCTTGTG-3' (SEQ ID NO: 2),

[0094] 5'-TGGTGAAAAAAAAAA-3'(SEQ ID NO:3)

[0095] Streptavidin solution concentration was 1 mg / mL; biotinylated DNA T The solution concentration is 100 μM, biotinylated DNA C The solution concentration was 100 μM.

[0096] Streptavidin and biotinylated DNA T and DNA C The volume ratio is 0.15:1.

[0097] (4) Assembly of coded test strips:

[0098] Use a pipette to absorb the above-mentioned spherical nucleic acid detection probe and evenly apply it to the gold label pad at a dosage of 8 μL / cm, and dry it at 37°C for 30 minutes; use a pen to dip the above-mentioned T-line probe and C-line probe respectively, and draw T lines and C lines on the nitrocellulose membrane respectively at a dosage of 0.5 μL / cm, and dry it at 37°C for 2 hours; immerse the sample pad in 50 mM Tris-HCl buffer and dry it at 37°C for 2 hours; assemble the base plate, sample pad, gold label pad, nitrocellulose membrane, and absorbent pad layer by layer, cut them into 3 mm wide test strips, and store them in a sealed bag at room temperature for later use.

[0099] Example 2: Investigation of the Effect of Spherical Nucleic Acid Detection Probe Structure Control on the Sensitivity of Nucleic Acid Test Strips

[0100] First, a comparative study was conducted on the salt aging method and the microwave heating method to investigate the sensitivity of the spherical nucleic acid detection probes prepared by the two methods when applied to the test strips. Figure 1 As shown in (a), a spherical nucleic acid detection probe is first prepared by salt aging and microwave heating. The prepared spherical nucleic acid detection probe can be used for the preparation of nucleic acid test strips. The C-line probe and T-line probe are fixed on the C-line and T-line of the nucleic acid test strip, respectively. When the target nucleic acid is present, one end of the target nucleic acid will first bind to the spherical nucleic acid detection probe (AuNPs-DNA) to form an AuNP-DNA-Target complex, which will continue to flow forward. Through base complementary pairing, the AuNPs-DNA-Target complex will bind to the DNA on the T line. T Combine to form AuNPs-DNA-Target-DNA TThe gold nanoparticles are fixed to the T line, and the T line appears red. The remaining spherical nucleic acid detection probes continue to flow forward and interact with the DNA on the C line. C Combination to form AuNPs-DNA-DNA C Complex, part of the gold nanoparticles are fixed to the C line, and the C line appears red. When the target nucleic acid does not exist, the spherical nucleic acid detection probe cannot form a complex on the T line, and the T line does not show color. The spherical nucleic acid detection probe is attached to the DNA on the C line. C The AuNPs-DNA-DNAc complex is formed and the C line is colored. When the target nucleic acid is present, the C line and the T line are colored at the same time. When the target nucleic acid is not present, only the C line is colored and the T line is not colored.

[0101] Spherical nucleic acid detection probes were prepared using the salt aging method and microwave heating method described in Example 1, respectively, and then prepared into test strips. A simulated product was prepared at a concentration of 0-100 nM and dripped onto the two test strips to examine the sensitivity of the spherical nucleic acid detection probes prepared by the two methods when applied to the test strips. The simulated product was a single-stranded DNA, the 5' end of which was complementary to the T-line probe and the 3' end of which was complementary to the polyA-DNA portion of the sequence attached to the spherical nucleic acid detection probe. The nucleotide sequence of the simulated product is shown in SEQ ID NO:14.

[0102] 5'-CACAAGCCTATCTATCAGCGCCTATGTCGATCTTACACTGTCATTC-3'(SEQ ID NO:14)

[0103] from Figure 1 (b) and Figure 1 The test results in (c) show that for the test strips prepared using the microwave heating method, the T-line color change is still quite obvious even at a simulated product concentration as low as 0.2 nM. In contrast, for the test strips prepared using the salt aging method, a slight color change in the T-line is only observed at a simulated product concentration of 5 nM. The corresponding detection limits for the two probes are 0.2 nM and 5 nM, respectively. This means that the spherical nucleic acid detection probes prepared using the microwave heating method have higher sensitivity in detection.

[0104] The microwave heating method is used to prepare spherical nucleic acid detection probes with a base length of 2-25. Figure 2 As shown in (a), the hydrated ion radius and zeta potential of spherical nucleic acid detection probes containing different A base lengths prepared by microwave heating and salt aging were measured, and the sensitivity of the test strips using different spherical nucleic acid detection probes was compared. Figure 2 (b) and (c) show that the number and density of spherical nucleic acid surface probes prepared by probes with different structures are different. Figure 2 As shown in (b), as the length of the A base sequence increases, the spherical nucleic acid hydrated ion radius shows an increasing trend, which may be due to the increase in probe length. Among them, the spherical nucleic acid hydrated ion radius corresponding to the A15 probe is slightly lower than that of A10, which may be due to the formation of secondary structure within the A15 probe sequence. Figure 2 As shown in (c), as the length of the A base sequence increases, the absolute value of the Zeta potential of the spherical nucleic acid becomes smaller. Because the gold nanoparticles themselves are negatively charged, and DNA is also negatively charged, the longer the A base sequence is, the fewer DNA probes are connected to the gold nanoparticles. This is related to the way poly A-DNA is connected to the gold nanoparticles. PolyA-DNA uses the strong adsorption force between the A base and the surface of the gold nanoparticle to achieve binding, and the surface area of ​​the gold nanoparticle is fixed. The more A bases there are, the larger the position they occupy on the surface of the gold nanoparticles, resulting in fewer DNA probes bound to the gold nanoparticles. We obtained the surface probe density of different spherical nucleic acids and the detection sensitivity of the test strips through experiments and calculations. As shown in Figure 2 As shown in (d), there are large differences in the probe density on the surface of different spherical nucleic acids. The more A bases there are, the smaller the probe density on the surface of the spherical nucleic acid is. In addition, there are also large differences in the detection sensitivity of the test strips constructed based on different spherical nucleic acids. Among them, the detection limit of the SH-SNAs encoded test strip is 5nM, the detection limit of the A2-SNAs encoded test strip is 5nM, the detection limit of the A5-SNAs encoded test strip is 1nM, the detection limit of the A7-SNAs encoded test strip is 0.5nM, the detection limit of the A10-SNAs encoded test strip is 0.2nM, the detection limit of the A15-SNAs encoded test strip is 0.5nM, the detection limit of the A20-SNAs encoded test strip is 10nM, and the detection limit of the A25-SNAs encoded test strip is 20nM. When the number of A bases is less than 10, as the number of A bases increases, the density of probes on the spherical nucleic acid surface decreases, and the detection limit of the test strip gradually decreases; when the number of A bases is greater than 10, as the number of A bases increases, the density of probes on the spherical nucleic acid surface decreases, and the detection limit of the test strip gradually increases; when the number of A bases is equal to 10, the detection limit of the test strip is the lowest and the sensitivity is the best.

[0105] In order to further explore the connection between AuNPs and DNA, SH-SNAs, A2-SNAs, A5-SNAs, A7-SNAs, A10-SNAs, A15-SNAs, A20-SNAs, A25-SNAs, and A10'-SNAs were first prepared. Among them, A10'-SNAs can be combined with spherical nucleic acid detection probes with A base lengths of 2-25 through Linker-DNA.

[0106] The polyA-DNA sequence used to prepare A10'-SNAs is A10'-DNA, the nucleotide sequence of which is shown in SEQ ID NO:15;

[0107] 5'-AAAAAAAAAATGCTGCACTGTCGACTCATTTTTTTTTTT-3'(SEQ ID NO:15)

[0108] 2 μL of 10 μM Linker-DNA and A10'-SNAs were mixed with spherical nucleic acid detection probes connected with DNA bases of different lengths and spherical nucleic acid detection probes prepared by salt aging method in PBS solution, and UV spectroscopy was performed to observe the changes of the UV absorption peak at 525 nm over time. The experimental results are shown in Figure 2. Figure 3 As shown in (a). Linker-DNA can connect A10'-SNAs with SH-SNAs, A2-SNAs, A5-SNAs, A7-SNAs, A10-SNAs, A15-SNAs, A20-SNAs, and A25-SNAs. The experimental results show that as the number of A bases increases and the reaction time prolongs, the ultraviolet peak red-shifts, and the ultraviolet absorption peak at 525nm decreases. This shows that the more A bases there are, the smaller the surface coverage of SNAs is, and the easier it is for Linker-DNA to bind to the two SNAs, resulting in a red shift of the ultraviolet peak; when the number of A bases gradually decreases from 25 to 2, the surface coverage of SNAs gradually increases, and due to the steric hindrance effect, linker-DNA is not easy to bind to the two SNAs, resulting in little change in the ultraviolet peak. The Linker-DNA nucleotide sequence is shown in SEQ ID NO: 16;

[0109] 5'-ACGTAGTCGCAACAGTGGTGTGAGTCGACAGTGCAGCA-3'(SEQ ID NO:16)

[0110] 2 μL of 10 μM fluorescent probe was mixed with spherical nucleic acid detection probes connected with DNA bases of different lengths and spherical nucleic acid detection probes prepared by salt aging method in PBS solution, and fluorescence spectrum scanning was performed to observe the change of fluorescence intensity over time. The experimental results are shown in Figure 2. Figure 3As shown in (c). The fluorescent probe can complement the DNA connected to the gold nanoparticles. When the fluorescent probe is combined with the spherical nucleic acid detection probe, the gold nanoparticles will quench the fluorescence of the fluorescent probe due to the Fluorescence Resonance Energy Transfer (FRET) effect. It can be found from the figure that as the number of A bases increases and the reaction time becomes longer, the fluorescence intensity gradually decreases. The experimental results show that the more A bases there are, the easier it is for the fluorescent probe to bind to the spherical nucleic acid detection probe, and the faster the fluorescence signal decreases. This shows that the more A bases there are on the probe, the less DNA is connected to the gold nanoparticles, and the steric hindrance is relatively small, and vice versa, which is consistent with the conclusions drawn above. The sequence of the fluorescent probe is shown in FAM-SEQ ID NO: 17 (5' end fluorescent modification group);

[0111] 5'-ATCACGTAGTCGCAACAGTGG-3'(SEQ ID NO:17)

[0112] To calculate the spacing between nucleic acid probes on the surface of gold nanoparticles, we first measured the probe concentrations in the supernatant before and after microwave heating, as well as the gold nanoparticle concentrations, and calculated the number of probes attached to each gold nanoparticle on different spherical nucleic acids. Next, assuming that the probes on the gold nanoparticles are evenly distributed on the sphere, we used the Fibonacci grid mathematical model to evenly distribute different numbers of points on the sphere. Assuming the radius of the sphere is 1, we took N points in total (for example, A10, we took 66 points). The coordinates of the nth point are (X n , Y n , Z n ), constant = (√5-1) / 2≈0.618, then the corresponding coordinate calculation formula is:

[0113] Z n =(2n-1) / N-1

[0114]

[0115] where φ is a constant.

[0116] like Figure 3 As shown in (c), select a point and calculate the straight-line distance between it and the four adjacent points based on the coordinates. Then, calculate the arc distance between the two points on the sphere using the cosine theorem and inverse trigonometric functions. The DNA spacing on the surface of spherical nucleic acid detection probes containing different A base lengths and spherical nucleic acid detection probes prepared by salt aging method is shown in Figure 2. Figure 3 As shown in (c).

[0117] Comparison revealed that the surface DNA spacing of spherical nucleic acid detection probes prepared by the salt aging method was the smallest, while the surface DNA spacing of spherical nucleic acid detection probes prepared by the microwave heating method increased with increasing number of A bases. Analysis of the sensitivity of test strips corresponding to spherical nucleic acid detection probes with different surface DNA spacings revealed that either too many or too few DNA connections resulted in reduced sensitivity. The optimal detection performance of nucleic acid test strips was achieved only when the surface coverage of gold nanoparticles was adequate. Specifically, as the number of A bases increased, the number of polyA-DNA probes attached to the surface of the spherical nucleic acid detection probe decreased, the surface probe spacing increased, and the steric hindrance to binding with the target nucleic acid decreased, facilitating the binding of the spherical nucleic acid to the target nucleic acid and the detection probe on the test strip. However, when the number of A bases was excessive, the number of polyA-DNA probes attached to the surface of the spherical nucleic acid detection probe was too small, resulting in a reduced probability of binding between the spherical nucleic acid and the target nucleic acid and the detection probe on the test strip, leading to poor detection performance. Only when the number of A bases was optimal (number of A bases = 10) could the lowest detection limit and optimal detection performance be achieved.

[0118] Example 3: Detection of novel coronavirus using nucleic acid amplification technology combined with nucleic acid test strips

[0119] Combine PCR amplification with coded test strips based on spherical nucleic acid detection probes to detect the new coronavirus. The detection principle and process are shown in Figure 4 (a) and Figure 4 In (d), the steps are as follows:

[0120] (1) Sample nucleic acid extraction:

[0121] The sample to be tested was mixed with lysis buffer (100 mM TCEP, 2 mM EDTA) at a ratio of 1:1, and lysed at room temperature for 5-10 min. The lysate was used for subsequent experiments.

[0122] (2) Amplify the target fragment:

[0123] The lysate was reverse transcribed, and the reverse transcription reaction system was as follows: 2 μL lysate, 0.5 μL RNase inhibitor (20 U / μL), 2 μL dNTP (2.5 mM), 1 μL reverse transcriptase (40 U / μL), 1 μL reverse primer (10 μM), 2 μL reverse transcriptase buffer (5×), DEPC water was added to 10 μL, and the mixture was incubated at 42°C for 1 h to obtain cDNA.

[0124] The cDNA was amplified by PCR. The PCR reaction system was as follows: 10 μL PCRMix (2×), 2 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and ddH2O was used to make up to 20 μL. The reaction procedure was denaturation at 95°C for 15 s, annealing and extension at 60°C for 1 min, and 40 cycles.

[0125] The forward primer is modified with ISP18 and has a hairpin structure at the 5' end; the forward primer is: 5'-SEQ ID NO:4 / ISP18 / SEQ ID NO:5-3';

[0126] 5'-AAGCCTCTTCTCGT-3'(SEQ ID NO:4)

[0127] 5'-ACGAGAAGAGGCTTGTGGTGACGGTAAAATGAAAGATCTCAGTC-3'(SEQ ID NO:5)

[0128] The reverse primer is modified with ISP18 and has a hairpin structure at the 5' end; the reverse primer is: 5'-SEQ ID NO: 6 / ISP18 / SEQ ID NO: 7-3';

[0129] 5'-TCACGTAGTCGCAA-3'(SEQ ID NO:6)

[0130] 5'-TTGCGACTACGTGACTGCCTGGAGTTGAATTTCTTGAACTGTTG-3' (SEQ ID NO: 7).

[0131] (3) Test strip detection and result reading:

[0132] The amplified product is added to the test strip for testing. If both the C and T lines of the test strip develop color, the test is positive; if only the C line develops color, the test is negative; if the C line does not develop color, the test strip is invalid.

[0133] The sensitivity of the detection method was investigated. SARS-CoV-2 pseudovirus (pseudovirus SARS-CoV-2-abEN, Order NO. M591001, purchased from Sangon Biotech (Shanghai) Co., Ltd.) was selected as the detection target. The amplified products of different target concentrations were added dropwise to the test strips, and the color development of the nucleic acid test strips was observed. The test results were as follows: Figure 4 As shown in (b), the detection limit is as low as 4 copies / reaction, and the negative logarithm of the concentration is fitted with the T / C grayscale value of the nucleic acid test strip, which also shows a good linear relationship.

[0134] The method was used to detect actual samples. The sample to be tested was mixed with a lysis buffer (100 mM TCEP, 2 mM EDTA) in a ratio of 1:1, and lysed at room temperature for 5-10 minutes to obtain a lysis product. The lysis product was reverse transcribed, and the reverse transcription reaction system was as follows: 2 μL of lysis product, 0.5 μL of RNase inhibitor (20 U / μL), 2 μL of dNTP (2.5 mM), 1 μL of reverse transcriptase (40 U / μL), 1 μL of reverse primer (10 μM), 2 μL of reverse transcriptase buffer (5×), DEPC water was added to 10 μL, and the mixture was incubated at 42°C for 1 hour to obtain cDNA. The cDNA was subjected to PCR reaction, and the PCR reaction system was as follows: 10 μL of PCRMix (2×), 2 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), add ddH2O to 20 μL, the reaction program is 95℃ denaturation for 15s, 60℃ annealing and extension for 1min, and 40 cycles; add PCR product to the test strip and read the test results. Figure 4 (e). Comparison of the detection results of this method with those of real-time fluorescence quantitative PCR method ( Figure 4 In (f), the test results were consistent with the real-time fluorescence quantitative PCR results, verifying the accuracy of the method combining nucleic acid amplification technology with nucleic acid test strips.

[0135] The target fragment sequence is shown in SEQ ID NO: 18, where N represents U:

[0136] 5'-NGGNANNNCNACNACCNAGGAACNGGGCCAGAAGCNGGACNNCCCNANGG NGCNAACAAAGACGGCNCCAAGGAACAACANNGCCAAAAGGCNNCNACGCAGAAGG GAGCAGAGGCGGCAGNCAAGCCNCNNCNCGNNCNCANCACGNAGNCGCAACAGN NCAAGAAANNCAACNCCAGGCAGCAGNAGGGGAACNNCNCCNGCNAGAANG-3'

[0137] (SEQ ID NO: 18)

[0138] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0139] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A coded test strip based on spherical nucleic acid detection probes, characterized in that: The spherical nucleic acid detection probe uses gold nanoparticles as its core, and poly A-DNA is immobilized on the surface of the gold nanoparticles by adsorption of poly A bases. The poly A-DNA consists of a single-stranded DNA and a poly A sequence at its 3' end. The sequence of the single-stranded DNA is shown in SEQ ID NO: 1, and the poly A has 10 bases. The spherical nucleic acid detection probe is prepared by mixing a gold nanoparticle solution and a poly A-DNA solution in a glass bottle, heating the glass bottle in a microwave for 5 minutes, adding ultrapure water for redissolution, centrifuging, and washing, repeating three times, and finally resuspending the precipitate in 1×PBS. The gold nanoparticles are prepared by a chloroauric acid-sodium citrate reduction method; the concentration of poly A-DNA in the poly A-DNA solution is 100 μM; and the volume ratio of the gold nanoparticle solution to the poly A-DNA solution is (10-20):

1.

2. A coded test strip based on spherical nucleic acid detection probes according to claim 1, characterized in that: The coded test strip is a universal nucleic acid detection test strip; it includes a base plate, and a sample pad, a gold label pad, a nitrocellulose membrane, and a water-absorbing pad that are sequentially overlapped with each other on the base plate. The gold label pad is coated with the spherical nucleic acid detection probe, and the nitrocellulose membrane is provided with a T line and a C line.

3. A coded test strip based on spherical nucleic acid detection probes according to claim 2, characterized in that: The sample pad is stacked on the gold mark pad at 1-2 mm, the gold mark pad is stacked on the nitrocellulose membrane at 1-2 mm, and the water absorbent pad is stacked on the nitrocellulose membrane at 1-2 mm.

4. A coded test strip based on spherical nucleic acid detection probes according to claim 2, characterized in that: The T-line is coated with streptavidin and 3'-end biotinylated DNA T , the C-line is coated with streptavidin and 3'-end biotinylated DNA C .

5. A coded test strip based on spherical nucleic acid detection probes according to claim 4, characterized in that: The DNA T The sequence is shown in SEQ ID NO: 2-Biotin, the DNA C The sequence is shown in SEQ ID NO: 3-Biotin.

6. The method for preparing a coded test strip based on a spherical nucleic acid detection probe according to any one of claims 1 to 5, characterized in that: The steps include: S1: Preparation of spherical nucleic acid detection probes; The gold nanoparticle solution and the poly A-DNA solution were mixed in a glass bottle, and the glass bottle was placed in a microwave oven and heated for 5 minutes. Ultrapure water was added to dissolve the mixture, and the mixture was centrifuged and washed three times. Finally, the precipitate was resuspended in 1×PBS to obtain the spherical nucleic acid detection probe. S2: Combine streptavidin with 3' biotinylated DNA T After incubation, the T-line probe was obtained and streptavidin was bound to the 3'-end biotinylated DNA. C After mixed incubation, the C-line probe was obtained; S3: Absorb the spherical nucleic acid detection probe and evenly apply it to the gold label pad; dip the T-line probe and C-line probe respectively, and draw T lines and C lines respectively on the nitrocellulose membrane; overlap the sample pad, gold label pad, nitrocellulose membrane, and absorbent pad on the bottom plate in sequence, and cut them to obtain the coded test strip.

7. The method for preparing a coded test strip based on spherical nucleic acid detection probes according to claim 6, wherein: In step S1, centrifugation was performed at 10,000 rpm for 10 min at 4°C, and washing was performed with 1× PBS; And / or, in step S1, the gold nanoparticles are prepared by a chloroauric acid-sodium citrate reduction method; and / or, in step S1, the concentration of poly A-DNA in the poly A-DNA solution is 100 μM; And / or, in step S1, the volume ratio of the gold nanoparticle solution to the poly A-DNA solution is (10-20):1; And / or, in step S2, the concentration of streptavidin is 1 mg / mL; the biotinylated DNA T The concentration was 100 μM, and the biotinylated DNA C The concentration was 100 μM; And / or, in step S2, the streptavidin binds to the biotinylated DNA T and DNA C The volume ratio is (0.1-2):1; and / or, in step S2, the incubation condition is incubating at 4°C for 1 hour; And / or, in step S3, the dosage of the spherical nucleic acid detection probe is 2-12 μL / cm, and the dosage of the T-line probe and the C-line probe are both 0.1-1 μL / cm.

8. A detection card, characterized in that: The test card includes a test strip and a card shell arranged on the outside of the test strip, the card shell includes an upper card shell and a lower card shell that are locked with each other, the lower card shell is provided with a card slot for placing the test strip, the upper card shell is provided with a sample addition port corresponding to the sample pad of the test strip, and is provided with an observation window corresponding to the nitrocellulose membrane of the test strip, and the T line and C line on the nitrocellulose membrane are both exposed in the observation window. The test strip is the test strip according to any one of claims 1 to 5 or the test strip prepared by the preparation method of claim 6 or 7.

9. A kit for detecting a novel coronavirus, characterized in that: include: The test strip according to any one of claims 1 to 5 or the test card according to claim 8; A forward primer, which is modified with ISP18 and has a hairpin structure at the 5' end; the forward primer is: 5'-SEQ ID NO:4 / ISP18 / SEQ ID NO:5-3'; A reverse primer is modified with ISP18 and has a hairpin structure at the 5' end; the reverse primer is: 5'-SEQ ID NO: 6 / ISP18 / SEQ ID NO: 7-3'.

10. A kit for detecting a novel coronavirus according to claim 9, characterized in that: The kit further comprises: Lysis buffer, which is a mixture of 100 mM TCEP and 2 mM EDTA in a volume ratio of 1:1; 20 U / μL RNase inhibitor, 2.5 mM dNTP, 40 U / μL reverse transcriptase, 5× reverse transcriptase buffer, 2× PCR Mix.

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