Encoding test strip based on spherical nucleic acid detection probe, kit and application

Abstract

The invention discloses a coding test strip based on a spherical nucleic acid detection probe, a kit and application, the spherical nucleic acid detection probe takes a gold nanoparticle as a core, polyA-DNA is adsorbed and fixed on the surface of the gold nanoparticle through a poly-A basic group; the polyA-DNA is composed of a single-stranded DNA and a poly A sequence at the 3'end of the single-stranded DNA, the sequence of the single-stranded DNA is as shown in SEQ ID NO: 1, and the number of bases of the poly A is 2-20. According to the spherical nucleic acid detection probe, DNA can be connected to the surface of the gold nanoparticles without additional modification through the adsorption effect of the gold nanoparticles on the A basic group, so that the preparation process of the spherical nucleic acid detection probe is simplified, and the detection cost is reduced; meanwhile, the detection sensitivity is improved by changing the length of poly-A and regulating and controlling the density and spacing of the spherical nucleic acid surface probe.

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 a spherical nucleic acid detection probe and applications thereof. Background Art

[0002] Fast and accurate disease diagnosis methods can save time, money and labor costs, improve medical efficiency, and reduce misdiagnosis rates and treatment costs. At present, the most commonly used screening test method for diseases caused by infectious pathogens is antigen test strips. Although antigen test strips have 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 test strips can significantly improve the sensitivity of pathogen detection. Currently used nucleic acid test strips are mainly divided into two types, the first type is a nucleic acid test strip based on antigen-antibody specific binding, and the second type is a nucleic acid test strip based on base complementary pairing. Nucleic acid test strips based on antigen-antibody specific binding may produce false positive signals due to primer dimers or nonspecific 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 single-base mutation detection. However, the current preparation methods of spherical nucleic acid probes used in this test strip, such as salt aging and freezing methods, require thiol modification of the nucleotide chains used, which takes a long time and is costly; this spherical nucleic acid constructed by chemical coupling has dense surface nucleic acid distribution, electrostatic repulsion and strong steric hindrance between adjacent oligonucleotides, resulting in a large number of recognition sites that cannot function, which may increase the difficulty of recognition and reduce the detection sensitivity. In addition, when used for nucleic acid testing, conventional nucleic acid amplification products are difficult to use directly with test strips and require high-temperature denaturation or uracil DNA glycosylase digestion 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 applications.

[0006] The technical solution adopted in the embodiment of the present invention is as follows:

[0007] First,

[0008] The embodiment of the present invention provides a coded test strip (for instant nucleic acid detection) based on a spherical nucleic acid detection probe. The spherical nucleic acid detection probe uses gold nanoparticles as the core, and polyA-DNA is fixed on the surface of the gold nanoparticles 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 bases of the poly A is 2-20.

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

[0010] The spherical nucleic acid detection probe on the coded test strip of the present invention takes gold nanoparticles as the core, and the polyA-DNA oligonucleotide chain is adsorbed on the surface of the gold nanoparticles through the polyA base; the present invention optimizes the density of the oligonucleotide chain adsorbed on the surface of the gold nanoparticles by regulating the length of the polyA, systematically adjusts the surface probe density and lateral spacing on the gold nanoparticles, and effectively improves the binding efficiency of the spherical nucleic acid detection probe with the object to be tested; the present invention adds a poly T sequence at the 5' end of the polyA-DNA, and utilizes the relatively weak binding force of the polyA-DNA with the gold nanoparticles to keep the adsorbed polyA-DNA in a relatively upright state, thereby promoting its binding with the T line probe and the C line probe. The middle sequence of the probe is a coding region, which is used to specifically identify and bind to the end sequence of the hairpin primer modified by ISP18.

[0011] Preferably, the number of bases of the poly A is 7-15, and 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 overlapped with each other in sequence on the base plate, wherein the gold label pad is coated with the spherical nucleic acid detection probe, and the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line).

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

[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 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 and improves the detection accuracy.

[0019] Second,

[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 for 5 minutes, and ultrapure water was added to dissolve, centrifuged, and washed, and the process was repeated three times. 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-labeled pad; dip the T-line probe and C-line probe respectively, and draw T-line and C-line respectively on the nitrocellulose membrane; overlap the sample pad, gold-labeled 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, centrifugation is performed at 4°C, 10,000 rpm for 10 minutes, and washing is performed with 1×PBS.

[0026] In some embodiments, in step S1, the gold nanoparticles are prepared by 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 and refluxing until boiling, quickly 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 is bound 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 further 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, wherein 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';

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

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

[0041] A reverse primer, wherein the reverse primer is modified by 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 of the kit of the present invention are modified by 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, can be used for real-time nucleic acid detection, and has good practicality.

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

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

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

[0048] Fifthly,

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

[0050] Sixth,

[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 sample to be tested; amplifying a target fragment in the sample to be tested by 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) Mix the sample to be tested with lysis buffer (100 mM TCEP, 2 mM EDTA) in a ratio of 1:1, and lyse at room temperature for 5-10 min. The lysate is 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 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 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;

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

[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 gold nanoparticles on A bases 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 spherical nucleic acid surface probes are regulated, thereby improving the detection sensitivity.

[0060] The present invention performs ISP18 modification on primers, and PCR products can be directly applied to coded test strips based on spherical nucleic acid detection probes, and the detection results are highly accurate and specific; the method has universality, and can be used for the detection of other viruses and pathogens only by adjusting the primer sequences accordingly; the test strips are used to output the detection results, and visual detection is achieved, which is user-friendly. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0062] Figure 1 The diagram is a comparison of the design principle and test results of the coded test strips, wherein (a) is a schematic diagram of the preparation of spherical nucleic acid detection probes by salt aging method and microwave heating method and their use in the construction of coded test strips, (b) is the sensitivity of the coded test strips based on poly A-DNA spherical nucleic acid detection probes prepared by microwave heating method; (c) is the sensitivity of the coded test strips based on spherical nucleic acid detection probes prepared by salt aging method;

[0063] Figure 2 The figure shows the characterization of spherical nucleic acid detection probes connected to polyA-DNA of different lengths and the detection sensitivity of the corresponding nucleic acid test strips, wherein (a) is a schematic diagram of gold nanoparticles connected to polyA-DNA of different lengths, (b) is a Zeta potential diagram 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 diagram of the surface probe density and the detection sensitivity;

[0064] Figure 3 The hybridization rate of spherical nucleic acid detection probes connected with polyA-DNA of different lengths and the characterization of the surface DNA spacing, wherein (a) is a graph showing the changes in the ultraviolet absorption spectra of spherical nucleic acid detection probes connected with polyA-DNA of different lengths and Linker-DNA at 525nm; (b) is a graph showing the changes in the fluorescence signal of the spherical nucleic acid detection probes connected with polyA-DNA of different lengths and the hybridization of the fluorescent probe; (c) is the calculated spacing of probes on the surface of gold nanoparticles connected with polyA-DNA of different lengths, and the right figure is a schematic diagram of the spacing; (d) is a schematic diagram showing the connection density of polyA-DNA of different lengths on gold nanoparticles;

[0065] Figure 4The detection principle of the spherical nucleic acid detection probe kit and the characterization of its sensitivity and specificity. (a) is a diagram of the detection principle of PCR combined with the coded test strip; (b) is the detection result of the coded test strip detecting different concentrations of SARS-CoV-2 pseudovirus; (c) is the standard curve of the gray value of the coded test strip determined by ImageJ; (d) is a schematic diagram of the SARS-CoV-2 virus detection process; (e) is the actual sample test strip detection result; (f) is the actual sample PCR detection result. DETAILED DESCRIPTION

[0066] 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 construed as limiting the present invention.

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

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

[0069] The variants of the new 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 a specific sub-range is explicitly stated.

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

[0072] Example 1: Microwave heating to synthesize spherical nucleic acid detection probes for the 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 with tin foil and store at 4°C away from light.

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

[0076] The spherical nucleic acid detection probe was prepared by microwave heating. 5 μL (100 μM) polyA-DNA solution and 100 μL gold nanoparticle solution were added to a 1.5 mL glass bottle and blown evenly. Then the glass bottle was placed in a microwave oven and heated at high temperature for 5 minutes, and then resuspended with ultrapure water. The solution was washed with 1× PBS, centrifuged at 10000 rpm for 10 minutes at 4°C, and the particles were resuspended in 1× PBS solution. The washing was repeated three times, and finally wrapped with tin foil and stored at 4°C away from light.

[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 is 2-25.

[0078] In Example 1 of the present invention, spherical nucleic acid detection probes containing different A base lengths are 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] The spherical nucleic acid detection probe was prepared by salt aging method, and the probe was named SH-SNAs (for comparison). 100 μL of gold nanoparticle solution, SH-DNA (100 μM, SEQ ID NO: 1 with SH modification at 3' end), and 5 μL of 10% (v / v) SDS were added to 89 μL of ultrapure water, mixed evenly, and placed in a 37°C water bath for 12 hours, and then 5M NaCl was added in batches (once every 2 hours, completed within 12 hours), and the spherical nucleic acid detection probe was obtained after incubation for 12 hours, which was wrapped in tin foil and stored at 4°C in the dark.

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

[0091] Separately bind streptavidin to 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 and 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 the 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 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 T line probe and C line probe respectively, and draw T line and C line 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 bottom 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 regulation on the sensitivity of nucleic acid test strips

[0100] Firstly, the salt aging method and microwave heating method were compared to investigate the sensitivity of the spherical nucleic acid detection probes prepared by the two methods when applied to 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, which 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 bound to the DNA on the C line. C The AuNPs-DNA-DNAc complex is formed by binding, 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 there is no target nucleic acid, only the C line is colored, and the T line is not colored.

[0101] The spherical nucleic acid detection probes were prepared by the salt aging method and microwave heating method described in Example 1, and prepared into test strips. The simulation 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 simulation product is a single-stranded DNA, whose 5' end is complementary to the T-line probe and whose 3' end is complementary to the polyA-DNA partial sequence connected to the spherical nucleic acid detection probe, and its nucleotide sequence is shown in SEQ ID NO: 14;

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

[0103] from Figure 1 (b) and Figure 1 From the test results in (c), we can see that for the test strips of spherical nucleic acid detection probes (A10-SNAs) prepared by microwave heating method, the T line color is still obvious when the simulated product concentration is as low as 0.2nM. However, for the test strips of spherical nucleic acid detection probes prepared by salt aging method, a slight color change can be observed on the T line only when the simulated product concentration is 5nM. The corresponding detection limits of the two probes are 0.2nM and 5nM, respectively, which means that the spherical nucleic acid detection probes prepared by 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 method and spherical nucleic acid detection probes prepared by salt aging method were measured, and the sensitivity of the test strips when 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 hydrated ion radius of the spherical nucleic acid shows an increasing trend, which may be due to the increase in the probe length. Among them, the hydrated ion radius of the spherical nucleic acid corresponding to the A15 probe is slightly lower than that of A10, which may be due to the formation of a secondary structure inside 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. Through experiments and calculations, we obtained the probe density on the surface of different spherical nucleic acids and the detection sensitivity of the test strips. As shown Figure 2 As shown in (d), there are large differences in the density of probes on the surfaces of different spherical nucleic acids. The more A bases there are, the smaller the density of probes on the surface of the spherical nucleic acid. In addition, there are also large differences in the detection sensitivity of test strips constructed based on different spherical nucleic acids. Among them, the detection limit of SH-SNAs coded test strip is 5nM, the detection limit of A2-SNAs coded test strip is 5nM, the detection limit of A5-SNAs coded test strip is 1nM, the detection limit of A7-SNAs coded test strip is 0.5nM, the detection limit of A10-SNAs coded test strip is 0.2nM, the detection limit of A15-SNAs coded test strip is 0.5nM, the detection limit of A20-SNAs coded test strip is 10nM, and the detection limit of A25-SNAs coded 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 surface of spherical nucleic acids 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 surface of spherical nucleic acids 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 which A10′-SNAs could be combined with spherical nucleic acid detection probes with an A base length of 2–25 via Linker-DNA.

[0106] The polyA-DNA sequence used to prepare A10'-SNAs is A10'-DNA, and its nucleotide sequence 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 A base DNA of different lengths and spherical nucleic acid detection probes prepared by salt aging method in PBS solution, and ultraviolet spectrum scanning was performed to observe the change of ultraviolet absorption peak at 525 nm over time. The experimental results are shown in 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, 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 A base DNA 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 as follows: Figure 3As shown in (c). The fluorescent probe can be complementary to 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 previously. 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 distance between nucleic acid probes on the surface of gold nanoparticles, we first measured the concentration of probes in the supernatant before and after microwave heating and the concentration of gold nanoparticles, and calculated the number of probes connected to each gold nanoparticle on different spherical nucleic acids. Next, assuming that the probes on the gold nanoparticles are evenly distributed on the spherical surface, we use the Fibonacci grid mathematical model to evenly distribute different numbers of points on the spherical surface. Assuming that the radius of the sphere is 1, we take N points in total (taking A10 as an example, we take 66 points), then 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 according to the coordinates. Then, calculate the arc distance between the two points on the sphere according to the cosine theorem and inverse trigonometric functions. The surface DNA spacing 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 3 As shown in (c).

[0117] By comparison, it was found that the surface DNA spacing of the spherical nucleic acid detection probe prepared by the salt aging method was the smallest, and the surface DNA spacing of the spherical nucleic acid detection probe prepared by the microwave heating method increased with the increase of the number of A bases. Combined with the analysis of the test strip detection sensitivity corresponding to the spherical nucleic acid detection probes with different surface DNA spacings, too much or too little DNA connection will lead to a decrease in sensitivity. Only when the surface coverage of gold nanoparticles is appropriate, the detection effect of the nucleic acid test strip can be best. That is, as the number of A bases increases, the number of polyA-DNA probes connected to the surface of the spherical nucleic acid detection probe decreases, the probe spacing on the surface of the gold nanoparticles increases, and the steric hindrance of binding with the target nucleic acid is reduced, which is conducive to the binding of the spherical nucleic acid with the target nucleic acid and the detection probe on the test strip. However, when the number of A bases is too large, the number of polyA-DNA probes connected to the surface of the spherical nucleic acid detection probe is too small, resulting in a decrease in the probability of binding between the spherical nucleic acid and the target nucleic acid and the detection probe on the test strip, so the detection effect is poor. Only when the number of A bases is appropriate (the number of A bases = 10) can the lowest detection limit be achieved and the best detection effect be achieved.

[0118] Example 3: Detection of novel coronavirus by combining nucleic acid amplification technology 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) Amplification of target fragment:

[0123] The cleavage product was reverse transcribed, and the reverse transcription reaction system was as follows: 2 μL cleavage product, 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 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), made up to 20 μL with ddH2O, and 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 by 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 by 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 dripped onto the test strip for detection. If both the C line and the T line of the test strip are colored, it is positive; if only the C line is colored, it is negative; if the C line is not colored, 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, and the amplification products of different target concentrations were dripped onto the test strips to observe the color development of the nucleic acid test strips. 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 solution (100 mM TCEP, 2 mM EDTA) in a ratio of 1:1, and lysed at room temperature for 5-10 min to obtain a lysis product. The lysis product was reverse transcribed, and the reverse transcription reaction system was as follows: 2 μL lysis product, 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. The cDNA was subjected to PCR reaction, and 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), add ddH2O to 20 μL, the reaction program is 95℃ denaturation for 15s, 60℃ annealing and extension for 1min, and cycle 40 times; add PCR product to the test strip and read the test result. Figure 4 (e). Compare the detection results of this detection 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" etc. 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0139] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary 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 has gold nanoparticles as the core, and polyA-DNA is fixed on the surface of the gold nanoparticles by adsorption of poly A bases. The poly A-DNA consists of 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 of the poly A is 2-20.

2. A coded test strip based on spherical nucleic acid detection probes according to claim 1, characterized in that: The poly A has 7-15 bases, and preferably, the poly A has 10 bases.

3. 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 comprises a bottom plate, and a sample pad, a gold label pad, a nitrocellulose membrane, and a water-absorbing pad which are overlapped with each other in sequence on the bottom plate, the gold label pad is coated with the spherical nucleic acid detection probe, and the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line); Optionally, the sample pad is stacked on the gold label pad at 1-2 mm, the gold label 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 3, characterized in that: 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 .

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 polyA-DNA solution were mixed in a glass bottle, and the glass bottle was placed in a microwave oven for 5 minutes, and ultrapure water was added to dissolve, centrifuged, and washed, and the process was repeated three times. Finally, the precipitate was resuspended in 1×PBS to obtain the spherical nucleic acid detection probe; 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; 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 line and C line 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, characterized in that: In step S1, centrifugation was performed at 4°C, 10,000 rpm for 10 min, and washing was performed with 1× PBS; And / or, in step S1, the gold nanoparticles are prepared by chloroauric acid-sodium citrate reduction method; and / or, in step S1, the concentration of polyA-DNA in the polyA-DNA solution is 100 μM; And / or, in step S1, the volume ratio of the gold nanoparticle solution to the polyA-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 is 100 μM; And / or, in step S2, the streptavidin and 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 detection 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, the detection line and the quality control line on the nitrocellulose membrane are exposed at the observation window, and 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 claims 1 to 5 or the test card according to claim 8; A forward primer, wherein 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'; A reverse primer, which is modified by 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 also includes: Lysis solution, which is a mixture of 100 mM TCEP and 2 mM EDTA in a volume ratio of 1:1; 20U / μL RNase inhibitor, 2.5mM dNTP, 40U / μL reverse transcriptase, 5× reverse transcriptase buffer, 2×PCRMix.

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