A reusable genotyping chip and detection method thereof

Abstract

The present invention belongs to the field of biochip technology, specifically a reusable genotyping chip and detection method thereof. The reusable genotyping chip provided by the present invention is prepared using polystyrene microspheres, capture probes, and a silicon substrate. During the SNP recognition stage, it relies on competitive DNA hybridization. The recognition sequence of the capture probe is converted into the target sequence itself, and the added detection probes are all hairpin probes with self-protection functions. This design significantly reduces the generation of false positive signals and significantly improves detection specificity. In addition, the entire process does not involve enzymes, which significantly simplifies the detection process and reduces detection costs.

Description

Technical Field

[0001] The present application relates to the field of biochip technology, and in particular to a reusable genotyping chip and detection technology thereof. Background Art

[0002] Biochip technology is a cutting-edge biotechnology that emerged in the 1980s. Using materials such as glass, silicon wafers, nylon membranes, or polymers as carriers, it integrates bioactive molecules at high density and utilizes molecular hybridization or immunoreaction principles to achieve high-throughput, multivariate biological detection and analysis. Based on the type of biomolecules immobilized on the chip, biochips can be broadly categorized into three main types: gene chips, protein chips, and tissue chips. Gene chips, a key branch of biochips, typically utilize microbeads or planar substrates as solid-phase supports. Specifically designed oligonucleotide probes are systematically immobilized on the carrier surface, allowing for specific hybridization with target nucleic acid molecules based on the principle of complementary base pairing. Hybridization results are acquired and analyzed using fluorescence imaging systems, ultimately enabling visual interpretation of biological information. With their high-throughput and highly parallel technology, gene chips have demonstrated significant value in areas such as gene expression profiling, mutation screening, and genomic polymorphism research.

[0003] Single nucleotide polymorphism (SNP), the most common form of genetic variation in the genome, refers to DNA sequence polymorphism caused by a single base change. Due to their widespread distribution and large number, SNPs have become important molecular markers in genetic research. Gene chip technology is naturally well-suited for SNP detection: the high frequency of SNPs requires a high-throughput detection platform, while gene chips offer the advantage of massively parallel detection. However, traditional solid-phase SNP detection chips, based on single-base extension or DNA ligation, are single-use, resulting in high testing costs and severely restricting the widespread application of this technology. To address this technical bottleneck, the development of reusable SNP genotyping chips and supporting detection technologies will be a key breakthrough in promoting the large-scale application of gene chip technology in genetic testing. Summary of the Invention

[0004] The present invention provides an erasable and regenerable single nucleotide polymorphism genotyping chip. The gene chip detection technology solves the problem in the prior art that solid-phase SNP gene detection chips can only be used once, resulting in high application costs.

[0005] A reusable genotyping chip is prepared using polystyrene microspheres, capture probes, and a silicon substrate. The primers used in conjunction with the chip include hairpin probe A, hairpin probe B, chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D; the capture probes include capture probe A and capture probe B.

[0006] Preferably, the sequence of the hairpin probe A comprises, from the 5' end to the 3' end, a stem sequence 1 of 10 to 16 bases, a loop sequence 1 of 10 to 16 bases, a stem sequence 2 of 10 to 16 bases and a chain hybridization trigger sequence 1 of 16 to 30 bases, wherein the stem sequence 1 and the loop sequence 1 are completely complementary to the wild-type target sequence, and form a mismatch with the mutant target sequence at the mutation site, and the mismatch site is located in the stem sequence 1.

[0007] Preferably, the sequence of the hairpin probe B includes, from the 5' end to the 3' end, a chain hybridization trigger sequence 2 of 16 to 30 bases, a stem sequence 3 of 10 to 16 bases, a loop sequence 2 of 10 to 16 bases and a stem sequence 4 of 10 to 16 bases, wherein the loop sequence 2 and the stem sequence 4 are completely complementary to the mutant target sequence, and form a mismatch with the wild-type target sequence at the mutation site, and the mismatch site is located in the stem sequence 4.

[0008] Preferably, the capture probe A comprises a transition sequence 1 of 0 to 15 bases and a capture sequence 1 of 10 to 16 bases from the 3' end to the 5' end, and the capture sequence 1 is completely or partially identical to the loop sequence 1 of the hairpin probe A.

[0009] Preferably, the capture probe B comprises a transition sequence 2 of 0 to 15 bases and a capture sequence 2 of 10 to 16 bases from the 5' end to the 3' end, and the capture sequence 2 is completely or partially identical to the loop sequence 2 of the hairpin probe B.

[0010] Preferably, the 5' ends of the fluorescent colorimetric probe A and the fluorescent colorimetric probe B are modified with the same fluorescent colorimetric group, and the sequence lengths of both are 20 to 60 bases.

[0011] Preferably, the 5' ends of the fluorescent colorimetric probe C and the fluorescent colorimetric probe D are modified with the same fluorescent colorimetric group, and the sequence lengths of both are 20 to 60 bases.

[0012] A detection method using a reusable genotyping chip is characterized in that the specific steps of the detection method are as follows:

[0013] S1: preparing and heating a reaction solution comprising hairpin probe A, hairpin probe B, and target DNA of a sample to be tested;

[0014] S2: Add the reaction solution dropwise onto the chip substrate loaded with capture probe A or capture probe B for hybridization reaction, and wash with buffer after the reaction is completed;

[0015] S3: Prepare a mixed probe solution 1 with chromogenic probes A and B, and drop it onto the cleaned chip substrate loaded with capture probe A for reaction; prepare a mixed probe solution 2 with chromogenic probes C and D, and drop it onto the cleaned chip substrate loaded with capture probe B for reaction, then wash with buffer and place under a fluorescence microscope for photography and recording. Single nucleotide polymorphism genotyping detection can be achieved based on the color of the fluorescence image;

[0016] S4: After use, the chip is incubated and cleaned by adding alkaline solution to erase the color probe. The erased chip can be used again according to the standard detection process.

[0017] Preferably, the pH of the alkaline solution in S4 is 12.

[0018] Preferably, the concentration of the hairpin probe A or B in S1 is 0.2-1 μM, and the heating is first heated at 85-95°C for 5-10 min, and then maintained at 0-10°C for 10-20 min.

[0019] Beneficial effects

[0020] The genotyping chip and its detection technology provided in this application have higher detection specificity compared to the existing ligase-based erasable chip technology. The technical feature of no enzyme participation in the entire process also greatly saves detection time and reduces detection costs.

[0021] Compared with the erasable chip based on ligase in the existing technology, the erasable chip based on ligase requires excessive addition of straight-chain primer probes A, B and C during the SNP recognition stage. In order to prevent false positive signals, the capture probe connected to the microbead needs to be used in combination with the blocking probe in advance. However, the inherent thermodynamic equilibrium characteristics of DNA hybridization make it impossible for the blocking probe to achieve a perfect full blocking function. The primer added in excess will still produce undesirable hybridization with the capture probe, thereby causing a false positive signal. The reusable chip of the present invention relies on competitive hybridization of DNA during the SNP recognition stage. The recognition sequence of the capture probe is cleverly converted into the target sequence itself, and the added detection probes are all hairpin probes with self-protection function. This design significantly reduces the generation of false positive signals and significantly improves the detection specificity. In addition, there is no enzyme involved in the whole process, which significantly simplifies the detection process and reduces the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the principle of the gene chip detection technology of this application;

[0023] Figure 2 This is a fluorescence microscopy image of the SNP1 genotyping test in Example 2 of the present application;

[0024] Figure 3 This is a fluorescence microscopy image of the SNP2 genotyping test in Example 3 of the present application;

[0025] Figure 4 This is a fluorescence microscopy image of the SNP3 genotyping test in Example 4 of the present application;

[0026] Figure 5 This is a fluorescence microscopy image of the SNP4 genotyping test in Example 5 of the present application;

[0027] Figure 6 This is a fluorescence microscopy image of the SNP5 genotyping test in Example 6 of the present application;

[0028] Figure 7 Fluorescence microscopy images of the gene chip erasure and reuse test in Example 7 of the present application;

[0029] Figure 8 This is a fluorescence microscopy image of the gene chip erasure and reuse test in Example 8 of the present application.

[0030] Figure 9 The following are technical comparison images of two gene chips for comparative example. DETAILED DESCRIPTION

[0031] Reagents used in the present invention:

[0032] PBST buffer (final concentration): 8 mM Na2HPO4, 2 mM KH2PO4, 10 mM KCl, 140 mM NaCl, 0.05% (v / v) Tween-20, pH 7.2-7.4.

[0033] 1× PBS buffer (final concentration): 10 mM Na2HPO4, 2 mM NaH2PO4, 135 mM NaCl, 4.7 mM KCl, pH 7.3±0.1.

[0034] Example 1. Preparation of a reusable genotyping chip.

[0035] 1. Prepare microbead solid phase chip substrate.

[0036] 1. Polystyrene microsphere activation step: Pre-prepared morpholineethanesulfonic acid buffer (MES, 0.1M, pH 6) and carboxyl polystyrene microspheres are prepared into a 5 mg / mL suspension. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution prepared using MES are added to the suspension until the concentrations of EDC and NHS in the suspension reach 25 mg / mL. After that, the suspension is shaken for 1 hour.

[0037] 2. Steps for covalently binding capture probes to polystyrene microspheres: Dissolve 10 nmol of dry powder of capture probe A or B to be linked in MES buffer, mix with 5 mg of activated polystyrene microsphere suspension, shake at room temperature for 4 hours, then wash three times by centrifugation with PBST buffer (5000 rpm, 5 min), and resuspend in ultrapure water to obtain a microbead solution in which polystyrene microspheres are covalently bound to capture probes A or B.

[0038] 3. A groove array region is formed on the first surface of a silicon substrate (10 mm × 10 mm × 1 mm) using photolithography.

[0039] 4. The polystyrene microspheres covalently bound to capture probes A or B were injected into ultrapure water and ultrasonically dispersed to prepare a monodisperse microbead solution with a concentration of 1 mg / mL. The etched silicon substrate was placed in a mounting groove (10 mm × 10 mm × 2 mm) in an assembly mold (75 mm × 25 mm × 3 mm). 150 μL of the monodisperse microbead solution was evenly dripped onto the first surface of the silicon substrate. A glass slide was covered on the assembly mold to ensure that there was no air in the liquid under the glass slide. After sealing with sealing film, the microbeads were placed face up in an ultrasonic cleaner and assembled using 120 W ultrasound for 10 min. The surface of the silicon substrate was cleaned twice with deionized water to complete the assembly of the microbeads and prepare a chip substrate loaded with capture probes.

[0040] 2. Design primers.

[0041] 1. Hairpin probe:

[0042] Hairpin probe A: From the 5' end to the 3' end, it includes a stem sequence 1 of 10 to 16 bases, a loop sequence 1 of 10 to 16 bases, a stem sequence 2 of 10 to 16 bases, and a strand hybridization trigger sequence 1 of 16 to 30 bases. The stem sequence 1 and loop sequence 1 of hairpin probe A are fully complementary to the wild-type target sequence and form a mismatch with the mutant target sequence at the mutation site. The mismatch site is located in the stem sequence 1.

[0043] From the 5' end to the 3' end, the hairpin probe B includes a chain hybridization trigger sequence 2 of 16 to 30 bases, a stem sequence 3 of 10 to 16 bases, a loop sequence 2 of 10 to 16 bases, and a stem sequence 4 of 10 to 16 bases. The loop sequence 2 and stem sequence 4 of the hairpin probe B are completely complementary to the mutant target, and form a mismatch with the wild-type target sequence at the mutation site. The mismatch site is located in the stem sequence 4.

[0044] 2. Capture probe:

[0045] The capture probe A includes, from the 3' end to the 5' end, a transition sequence 1 of 0 to 15 bases and a capture sequence 1 of 10 to 16 bases, wherein the capture sequence 1 is completely identical or partially identical to the loop sequence 1 of the hairpin probe A;

[0046] The capture probe B includes a transition sequence 2 of 0 to 15 bases and a capture sequence 2 of 10 to 16 bases from the 5' end to the 3' end. The capture sequence 2 is completely or partially identical to the loop sequence 2 of the hairpin probe B.

[0047] 3. Fluorescent colorimetric probe:

[0048] The fluorescent colorimetric probes include fluorescent colorimetric probe A, fluorescent colorimetric probe B, fluorescent colorimetric probe C and fluorescent colorimetric probe D, wherein the 5' ends of fluorescent colorimetric probe A and fluorescent colorimetric probe B are modified with the same fluorescent colorimetric group, and the 5' ends of fluorescent colorimetric probe C and fluorescent colorimetric probe D are modified with the same fluorescent colorimetric group, and the sequence lengths are all 20 to 60 bases; all fluorescent colorimetric probes can self-fold to form hairpin structures, wherein the chain hybridization trigger sequence 2 of the hairpin probe B can trigger the alternating ring-opening self-assembly of the hairpins of the fluorescent colorimetric probe A and the fluorescent colorimetric probe B, and the chain hybridization trigger sequence 1 of the hairpin probe A can trigger the alternating ring-opening self-assembly of the hairpins of the fluorescent colorimetric probe C and the fluorescent colorimetric probe D.

[0049] Example 2. Detection of SNP1.

[0050] 1. Target sequences and probes, as shown in Table 1.

[0051] Table 1

[0052]

[0053] 2. Target sequence comparison test.

[0054] S1: Prepare 100 μL of reaction solution using 1× PBS buffer. The reaction solution includes: 50 nM target sequence (mutant target sequence or wild-type target sequence), 0.5 μM hairpin probe A, and 0.5 μM hairpin probe B. Execute the temperature program: heat at 95°C for 5 minutes and hold at 4°C for 20 minutes to obtain a mixed solution after the reaction.

[0055] S2: The mixed solution after the reaction was added dropwise onto the chip substrate loaded with the capture probes, and the hybridization reaction was carried out at 25°C for 30 minutes. The mixture was then gently washed three times with 1×PBST buffer.

[0056] S3: Prepare chromogenic probe A, B, C, and D solutions in 1× PBST buffer at a concentration of 2 μM each. Heat each solution at 95°C for 5 min and allow to cool to room temperature. Prepare a mixed probe solution 1 (0.5 μM each) of chromogenic probes A and B and a mixed probe solution 2 (0.5 μM each) of chromogenic probes C and D for later use.

[0057] S4: Add freshly prepared mixed probe solution 1 to the chip substrate loaded with capture probe A after cleaning in S1, and add freshly prepared mixed probe solution 2 to the chip substrate loaded with capture probe B after cleaning. React at 25°C for 30 min, wash lightly with 1×PBST buffer solution three times, and place under a fluorescence microscope to record the fluorescence microscopic images of Cy5 and FAM, respectively.

[0058] like Figure 2 As shown, for mutant targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B did not emit light under the FAM fluorescence channel; for wild-type targets, the microbeads on the chip loaded with capture probe A did not emit light under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B exhibited obvious fluorescence under the FAM fluorescence channel; for heterozygous (mutant + wild) targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B also exhibited obvious fluorescence under the FAM fluorescence channel; the experimental results were consistent with the expected results, indicating that this chip can be used for SNP1 genotyping detection and analysis.

[0059] Example 3. Detection of SNP2.

[0060] 1. Target sequences and probes, as shown in Table 2.

[0061] Table 2

[0062]

[0063] 2. Target sequence comparison test.

[0064] The difference from Example 2 is:

[0065] 1. The concentration of hairpin probes A and B is 0.4 μM. Execute the temperature program: heat at 90°C for 10 min, hold at 4°C for 20 min, and obtain a mixed solution after reaction.

[0066] 2. The mixed solution after reaction was hybridized on the chip substrate at 20°C for 40 minutes.

[0067] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 1 μM.

[0068] 4. Add mixed probe solution 1 or mixed probe solution 2 onto the chip substrate and react at 30°C for 25 min.

[0069] like Figure 3 As shown, for mutant targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B did not emit light under the FAM fluorescence channel; for wild-type targets, the microbeads on the chip loaded with capture probe A did not emit light under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B exhibited obvious fluorescence under the FAM fluorescence channel; for heterozygous (mutant + wild) targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B also exhibited obvious fluorescence under the FAM fluorescence channel; the experimental results were consistent with the expected results, indicating that this chip can be used for SNP2 genotyping detection and analysis.

[0070] Example 4. Detection of SNP3.

[0071] 1. Target sequences and probes, as shown in Table 3.

[0072] Table 3

[0073]

[0074] 2. Target sequence comparison test.

[0075] The difference from Example 2 is:

[0076] 1. The concentration of hairpin probes A and B is 0.8 μM. A temperature program is performed: heating at 85°C for 10 min and holding at 8°C for 15 min to obtain a mixed solution after reaction.

[0077] 2. The mixed solution after reaction was hybridized on the chip substrate at 35°C for 20 minutes.

[0078] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 1.5 μM.

[0079] 4. Add mixed probe solution 1 or mixed probe solution 2 onto the chip substrate and react at 20°C for 10 min.

[0080] like Figure 4As shown, for mutant targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B did not emit light under the FAM fluorescence channel; for wild-type targets, the microbeads on the chip loaded with capture probe A did not emit light under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B exhibited obvious fluorescence under the FAM fluorescence channel; for heterozygous (mutant + wild) targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B also exhibited obvious fluorescence under the FAM fluorescence channel; the experimental results were consistent with the expected results, indicating that this chip can be used for SNP3 genotyping detection and analysis.

[0081] Example 5. Detection of SNP4.

[0082] 1. Target sequences and probes, as shown in Table 4.

[0083] Table 4

[0084]

[0085] 2. Target sequence comparison test.

[0086] The difference from Example 2 is:

[0087] 1. The concentration of hairpin probes A and B is 0.2 μM. Execute the temperature program: heat at 95°C for 10 min, then hold at 0°C for 10 min.

[0088] 2. The mixed solution after reaction was hybridized on the chip substrate at 20°C for 20 min.

[0089] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 2 μM.

[0090] 4. Add mixed probe solution 1 or mixed probe solution 2 onto the chip substrate and react at 30°C for 60 min.

[0091] like Figure 5As shown, for mutant targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B did not emit light under the FAM fluorescence channel; for wild-type targets, the microbeads on the chip loaded with capture probe A did not emit light under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B exhibited obvious fluorescence under the FAM fluorescence channel; for heterozygous (mutant + wild) targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B also exhibited obvious fluorescence under the FAM fluorescence channel; the experimental results were consistent with the expected results, indicating that this chip can be used for SNP4 genotyping detection and analysis.

[0092] Example 6. Detection of SNP5.

[0093] 1. Target sequences and probes, as shown in Table 5.

[0094] Table 5

[0095]

[0096] 2. Target sequence comparison test.

[0097] The difference from Example 2 is:

[0098] 1. The concentration of hairpin probes A and B is 1.0 μM. Execute the temperature program: heat at 90°C for 5 min, then hold at 10°C for 20 min to obtain a mixed solution after reaction.

[0099] 2. The mixed solution after reaction was hybridized on the chip substrate at 40°C for 20 minutes.

[0100] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 0.8 μM.

[0101] 4. Add mixed probe solution 1 or mixed probe solution 2 onto the chip substrate and react at 40°C for 30 min.

[0102] like Figure 6As shown, for mutant targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B did not emit light under the FAM fluorescence channel; for wild-type targets, the microbeads on the chip loaded with capture probe A did not emit light under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B exhibited obvious fluorescence under the FAM fluorescence channel; for heterozygous (mutant + wild) targets, the microbeads on the chip loaded with capture probe A exhibited obvious fluorescence under the Cy5 fluorescence channel, while the microbeads on the chip loaded with capture probe B also exhibited obvious fluorescence under the FAM fluorescence channel; the experimental results were consistent with the expected results, indicating that this chip can be used for SNP5 genotyping detection and analysis.

[0103] Example 7. Chip erasure and reuse test 1.

[0104] The target sequence mutant, hairpin probe A, hairpin probe B, capture probe A, capture probe B, chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D are the same as those in Example 2.

[0105] The mutant target sequence test was the same as in Example 2.

[0106] Alkaline washing of microbead solid phase chip substrate: After use, add alkaline solution with pH 12 and incubate at room temperature for three times, each time for 10 minutes, and then wash with 1× PBST until neutral.

[0107] The mutant target sequence test and microbead solid phase chip substrate alkaline washing steps were repeated 10 times.

[0108] The results are as follows Figure 7 As shown, after each use, the gene chip can be effectively erased by washing with alkaline solution, removing the hybridization probe and extinguishing the fluorescence. It can function normally again and accurately identify mutant target sequences, showing good reusability.

[0109] Example 8. Chip erasure and reuse test 2.

[0110] The target sequence wild type, hairpin probe A, hairpin probe B, capture probe A, capture probe B, chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D are the same as those in Example 2.

[0111] The wild-type target sequence test was the same as in Example 2.

[0112] Alkaline cleaning of the microbead solid phase chip substrate: After use, the chip was incubated and cleaned three times with an alkaline solution of pH 12 at room temperature for 10 min each time, and then washed with 1× PBST until neutral.

[0113] The above-mentioned wild-type target sequence test and microbead solid phase chip substrate alkaline washing steps were repeated 10 times.

[0114] The results are as follows Figure 8 As shown, after each use, the gene chip can be effectively erased by washing with alkaline solution, removing the hybridization probe and extinguishing the fluorescence. It can function normally again and accurately identify wild-type target sequences, showing good reusability.

[0115] Comparative Example. This application is compared with the erasable chip technology based on ligase.

[0116] The target sequence mutant and wild type were the same as in Example 2, and the target sequence was compared and tested using the currently invented microbead chip technology and the ligase-based erasable chip technology (CN 119193793 A).

[0117] The microbead chip technology testing method and probe design of the present invention are the same as those in Example 2.

[0118] The test method and probe design of the ligase-based erasable chip technology are shown in Table 6. The chip preparation and detection scheme refer to CN 119193793 A:

[0119] Table 6

[0120]

[0121] Comparative test results such as Figure 9 As shown, the detection specificity of the technology of the present invention is significantly better in distinguishing the same mutant target or wild-type target, which is specifically manifested in lower false positive signals and higher discrimination factors (discrimination factor = true signal / false positive signal). Figure 9 The specific values ​​are shown in Table 7:

[0122] Table 7

[0123]

Claims

1. A reusable genotyping chip, characterized in that: The chip is prepared by polystyrene microspheres, capture probes and a silicon substrate. The primers used in conjunction with the chip include hairpin probe A, hairpin probe B, fluorescent colorimetric probe A, fluorescent colorimetric probe B, fluorescent colorimetric probe C and fluorescent colorimetric probe D; the capture probes include capture probe A and capture probe B; The sequence of the hairpin probe A comprises, from the 5' end to the 3' end, a stem sequence 1 of 10 to 16 bases, a loop sequence 1 of 10 to 16 bases, a stem sequence 2 of 10 to 16 bases, and a strand hybridization trigger sequence 1 of 16 to 30 bases, wherein the stem sequence 1 and the loop sequence 1 are completely complementary to the wild-type target sequence, and form a mismatch with the mutant target sequence at the mutation site, and the mismatch site is located in the stem sequence 1; The sequence of the hairpin probe B includes, from the 5' end to the 3' end, a strand hybridization trigger sequence 2 of 16 to 30 bases, a stem sequence 3 of 10 to 16 bases, a loop sequence 2 of 10 to 16 bases, and a stem sequence 4 of 10 to 16 bases, wherein the loop sequence 2 and the stem sequence 4 are completely complementary to the mutant target and form a mismatch with the wild-type target sequence at the mutation site, and the mismatch site is located in the stem sequence 4; The capture probe A includes a transition sequence 1 of 0 to 15 bases and a capture sequence 1 of 10 to 16 bases from the 3' end to the 5' end, and the capture sequence 1 is completely or partially identical to the loop sequence 1 of the hairpin probe A; The capture probe B includes a transition sequence 2 of 0 to 15 bases and a capture sequence 2 of 10 to 16 bases from the 5' end to the 3' end, and the capture sequence 2 is completely or partially identical to the loop sequence 2 of the hairpin probe B; The 5' ends of the fluorescent colorimetric probe A and the fluorescent colorimetric probe B are modified with the same fluorescent colorimetric group, and the sequence lengths of both are 20 to 60 bases; The 5' ends of the fluorescent colorimetric probes C and D are modified with the same fluorescent colorimetric group, and the sequence lengths of both are 20 to 60 bases; The fluorescent colorimetric probe A, fluorescent colorimetric probe B, fluorescent colorimetric probe C and fluorescent colorimetric probe D can all self-fold to form hairpin structures, wherein the chain hybridization trigger sequence 2 of the hairpin probe B can trigger the alternating ring-opening self-assembly of the hairpins of the fluorescent colorimetric probe A and the fluorescent colorimetric probe B, and the chain hybridization trigger sequence 1 of the hairpin probe A can trigger the alternating ring-opening self-assembly of the hairpins of the fluorescent colorimetric probe C and the fluorescent colorimetric probe D.

2. A detection method using the reusable genotyping chip according to claim 1 for purposes other than disease diagnosis and treatment, characterized in that: The specific steps of the detection method are as follows: S1: preparing a reaction solution and heating it, wherein the reaction solution includes hairpin probe A, hairpin probe B and target DNA of the sample to be tested; S2: adding the reaction solution dropwise onto the chip substrate loaded with capture probe A and the chip substrate loaded with capture probe B to perform hybridization reaction, and washing with buffer after the reaction is completed; S3: preparing a mixed probe solution 1 of fluorescent colorimetric probes A and B, and adding it dropwise to the cleaned chip substrate loaded with capture probe A for reaction; Fluorescent colorimetric probes C and D are prepared into a mixed probe solution 2, which is then added dropwise to the cleaned chip substrate loaded with capture probe B for reaction. The solution is then washed with a buffer solution and photographed under a fluorescence microscope. Single nucleotide polymorphism genotyping can be achieved based on the color of the fluorescent image. S4: After use, the chip is incubated and cleaned by adding alkaline solution to erase the fluorescent colorimetric probe. The erased chip can be used again according to the standard detection process.

3. The detection method according to claim 2, characterized in that The pH of the alkaline solution in S4 is 12.

4. The detection method according to claim 2, characterized in that: The concentration of the hairpin probe A or B in S1 is 0.2-1 μM, and the heating is first heated at 85-95°C for 5-10 min, and then maintained at 0-10°C for 10-20 min.

Images