Reusable genotyping chip and detection method thereof

Abstract

The invention belongs to the technical field of biochips, and particularly relates to a reusable genetic typing chip and a detection method thereof, the reusable genetic typing chip provided by the invention is prepared from polystyrene microspheres, capture probes and a silicon substrate, and depends on competitive hybridization of DNA in an SNP recognition stage, and the detection result is more accurate. The recognition sequence of the capture probe is converted into the sequence of the target object, and the added detection probes are hairpin probes with a self-protection function, so that the generation of false positive signals is remarkably reduced, and the detection specificity is remarkably improved. And no enzyme participates in the whole process, so that the detection process is remarkably simplified, and the detection cost is reduced.

Description

Technical Field

[0001] This application relates to the field of biochip technology, and particularly to a reusable genotyping chip and its detection technology. Background Art

[0002] Biochip technology is a cutting-edge biotechnology that emerged in the 1980s. This technology uses materials such as glass, silicon wafers, nylon membranes, or polymer matrices as carriers, integrates bioactive molecules with high density, and realizes high-throughput multi-biological detection and analysis based on the principles of molecular hybridization or immune reactions. According to the types of bio-molecules immobilized on the chip, biochips can be mainly divided into three categories: gene chips, protein chips, and tissue chips. Among them, as an important branch of biochips, gene chips usually use microbeads or planar matrices as solid-phase carriers, orderly immobilize specifically designed oligonucleotide probes on the surface of the carrier, and perform specific hybridization with target nucleic acid molecules based on the principle of base complementary pairing. The hybridization results are collected and analyzed through a fluorescence imaging system, and finally the visualization interpretation of biological information is realized. With its high-throughput and high-parallel technical advantages, gene chips have shown important value in fields such as gene expression profiling analysis, mutation screening, and genomic polymorphism research.

[0003] Single Nucleotide Polymorphism (SNP), as the most common form of genetic variation in the genome, refers to the phenomenon of DNA sequence polymorphism caused by single-base variation. Due to its wide distribution and large quantity, SNP has become an important molecular marker in genetic research. Gene chip technology and SNP detection have natural adaptability: on the one hand, the high frequency of SNP requires a high-throughput detection platform, and on the other hand, gene chips happen to have the advantage of large-scale parallel detection. However, traditional solid-phase SNP detection chips are based on the principles of single-base extension or DNA ligation, and their "single-use" characteristic leads to high detection costs, severely restricting the popularization and application of this technology. To address this technical bottleneck, the development of reusable SNP genotyping chips and supporting detection technologies will become the key breakthrough point for promoting the large-scale application of gene chip technology in the field of genetic detection. Summary of the Invention

[0004] The present invention provides an erasable and regenerable single nucleotide polymorphism genotyping chip, and the detection technology of this gene chip 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, which is obtained by preparing 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 sequentially includes a stem sequence 1 of 10-16 bases, a loop sequence 1 of 10-16 bases, a stem sequence 2 of 10-16 bases, and a strand hybridization trigger sequence 1 of 16-30 bases from the 5' end to the 3' end. The stem sequence 1 and the loop sequence 1 are completely complementary to the wild-type target sequence and form a mismatch at the mutation site with the mutant target sequence, and the mismatch site is located in the stem sequence 1.

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

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

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

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

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

[0012] A detection method using a reusable genotyping chip, characterized in that the specific steps of the detection method are as follows: S1: Prepare a reaction solution and heat it. The reaction solution includes hairpin probe A, hairpin probe B and the target DNA of the sample to be tested. S2: Drop the reaction solution onto the chip substrate loaded with capture probe A or capture probe B for hybridization reaction, and after the reaction is completed, wash it with a buffer solution. S3: Prepare the color - developing probes A and B into a mixed probe solution 1, and drop it onto the washed chip substrate loaded with the capture probe A for reaction; prepare the color - developing probes C and D into a mixed probe solution 2, and drop it onto the washed chip substrate loaded with the capture probe B for reaction. Then, wash it with a buffer solution and place it under a fluorescence microscope for photographing and recording. The single - nucleotide polymorphism genotyping detection can be achieved according to the color of the fluorescence image; S4: Drop an alkaline solution onto the used chip for incubation and washing to achieve the erasure of the color - developing probe. The erased chip can be reused according to the standard detection process.

[0013] Preferably, the pH of the alkaline solution in S4 is 12.

[0014] Preferably, the concentration of the hairpin probe A or B in S1 is 0.2 - 1 μM. The heating is first at 85 - 95 °C for 5 - 10 min, and then maintained at 0 - 10 °C for 10 - 20 min.

[0015] Beneficial effects The genotyping chip and its detection technology provided by this application have higher detection specificity compared with the existing ligase - based erasable chip technology. The technical feature of no enzyme participation throughout the process also greatly saves the detection time and reduces the detection cost.

[0016] Compared with the existing ligase - based erasable chip in the prior art, in the SNP recognition stage of the ligase - based erasable chip, an excessive amount of linear primer probes A, B, and C need to be added. To prevent false - positive signals, the capture probes connected to the microbeads need to be used in combination with blocking probes in advance. However, due to the inherent thermodynamic equilibrium characteristics of DNA hybridization, the blocking probes cannot achieve a perfect full - blocking function, and the excessive primers added will still hybridize with the capture probes undesirably, thus causing false - positive signals. In contrast, the reusable chip of the present invention relies on competitive hybridization of DNA in the SNP recognition stage. The recognition sequence of the capture probe is ingeniously transformed into the target sequence itself, and the detection probes added are all hairpin probes with self - protection functions. This design significantly reduces the generation of false - positive signals and significantly improves the detection specificity. And there is no enzyme participation throughout the process, which significantly simplifies the detection process and reduces the detection cost. Brief description of the drawings

[0017] Figure 1 It is a schematic diagram of the principle of the gene chip detection technology of this application; Figure 2 It is a fluorescence microscopic image of the SNP1 genotyping detection in Example 2 of this application; Figure 3 It is a fluorescence microscopic image of the SNP2 genotyping detection in Example 3 of this application; Figure 4 It is the fluorescence microscopic image of SNP3 genotyping detection for Example 4 of this application; Figure 5 It is the fluorescence microscopic image of SNP4 genotyping detection for Example 5 of this application; Figure 6 It is the fluorescence microscopic image of SNP5 genotyping detection for Example 6 of this application; Figure 7 It is the fluorescence microscopic image of gene chip erasure and reuse test for Example 7 of this application; Figure 8 It is the fluorescence microscopic image of gene chip erasure and reuse test for Example 8 of this application.

[0018] Figure 9 It is the technical comparison image of two gene chips for the comparative example. Specific implementation mode

[0019] Reagents used in the present invention: 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.

[0020] 1×PBS buffer (final concentration): 10 mM Na2HPO4, 2 mM NaH2PO4, 135 mM NaCl, 4.7 mM KCl, pH 7.3 ± 0.1.

[0021] Example 1. Preparation of reusable genotyping chip.

[0022] I. Preparation of microbead solid-phase chip substrate.

[0023] 1. Polystyrene microsphere activation step: Prepare a suspension of 5 mg / mL by mixing the pre-prepared morpholine ethanesulfonic acid buffer (MES, 0.1M, pH 6) with carboxyl polystyrene microspheres; then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution prepared with MES, and after the concentrations of EDC and NHS in the suspension system both reach 25 mg / mL, shake for 1 h.

[0024] 2. Steps for covalently binding capture probes to polystyrene microspheres: After dissolving 10 nmol of dry powder of capture probe A or B to be ligated in MES buffer, it is mixed with the activated polystyrene microsphere suspension, where the content of polystyrene microspheres is 5 mg. React with shaking at room temperature for 4 hours, then centrifuge and wash three times with PBST buffer (5000 rpm, 5 min), and resuspend with ultrapure water to obtain a microsphere solution in which polystyrene microspheres are covalently bound with capture probe A or B.

[0025] 3. Use photolithography to form a groove array region on the first surface of a silicon substrate (10 mm × 10 mm × 1 mm).

[0026] 4. Inject the above microspheres in which polystyrene microspheres are covalently bound with capture probe A or B into ultrapure water, and prepare a monodisperse microsphere solution with a concentration of 1 mg / mL through ultrasonic dispersion; place the etched silicon substrate into the installation groove (10 mm × 10 mm × 2 mm) in an assembly mold (75 mm × 25 mm × 3 mm), evenly drop 150 μL of the monodisperse microsphere solution on the first surface of the silicon substrate, cover a glass slide on the assembly mold, and ensure that there is no air in the liquid under the glass slide. After sealing with a sealing film and winding, place it face up in an ultrasonic cleaner, and perform ultrasonic assembly at 120 W for 10 min. Wash the surface of the silicon substrate twice with deionized water to complete the assembly of the microspheres and prepare a chip substrate loaded with capture probes.

[0027] II. Design primers.

[0028] 1. Hairpin probes: Hairpin probe A: Sequentially includes a stem sequence 1 of 10 - 16 bases, a loop sequence 1 of 10 - 16 bases, a stem sequence 2 of 10 - 16 bases, and a strand hybridization trigger sequence 1 of 16 - 30 bases from the 5' end to the 3' end. The stem sequence 1 and the loop sequence 1 of hairpin probe A are completely complementary to the wild-type target sequence and form a mismatch at the mutation site with the mutant target sequence, and the mismatch site is located in the stem sequence 1; Hairpin probe B: Sequentially includes a strand hybridization trigger sequence 2 of 16 - 30 bases, a stem sequence 3 of 10 - 16 bases, a loop sequence 2 of 10 - 16 bases, and a stem sequence 4 of 10 - 16 bases from the 5' end to the 3' end. The loop sequence 2 and the stem sequence 4 of hairpin probe B are completely complementary to the mutant target and form a mismatch at the mutation site with the wild-type target sequence, and the mismatch site is located in the stem sequence 4.

[0029] 2. Capture probes: The capture probe A sequentially includes a transition sequence 1 of 0 to 15 bases from the 3'-end to the 5'-end, and a capture sequence 1 of 10 to 16 bases. The capture sequence 1 is exactly the same as or partially the same as the loop sequence 1 of the hairpin probe A. The capture probe B sequentially includes a transition sequence 2 of 0 to 15 bases from the 5'-end to the 3'-end, and a capture sequence 2 of 10 to 16 bases. The capture sequence 2 is exactly the same as or partially the same as the loop sequence 2 of the hairpin probe B.

[0030] 3. Fluorescent chromogenic probe: The fluorescent chromogenic probe includes fluorescent chromogenic probe A, fluorescent chromogenic probe B, fluorescent chromogenic probe C, and fluorescent chromogenic probe D. The 5'-ends of fluorescent chromogenic probe A and fluorescent chromogenic probe B are modified with the same fluorescent chromogenic group, and the 5'-ends of fluorescent chromogenic probe C and fluorescent chromogenic probe D are modified with the same fluorescent chromogenic group. The sequence length of each is 20 to 60 bases; all fluorescent chromogenic probes can self-fold into hairpin structures. Among them, the strand hybridization trigger sequence 2 of the hairpin probe B can trigger the hairpin alternating ring-opening self-assembly of fluorescent chromogenic probe A and fluorescent chromogenic probe B, and the strand hybridization trigger sequence 1 of the hairpin probe A can trigger the hairpin alternating ring-opening self-assembly of fluorescent chromogenic probe C and fluorescent chromogenic probe D.

[0031] Example 2. Detecting SNP1.

[0032] I. Target sequence and probes, as shown in Table 1.

[0033] Table 1

[0034] II. Target sequence comparative test.

[0035] S1: Prepare 100 μL of reaction solution with 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 min, and maintain at 4 °C for 20 min to obtain the reaction mixture.

[0036] S2: Drop the reaction mixture on the chip substrate loaded with the capture probe, perform hybridization reaction at 25 °C for 30 min, and gently wash 3 times with 1×PBST buffer.

[0037] S3: Prepare solutions of chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D with 1×PBST buffer respectively, with a concentration of 2 μM each. Heat at 95 °C for 5 min respectively, and cool to room temperature naturally. Then prepare a mixed probe solution 1 of chromogenic probe A and B with a concentration of 0.5 μM each, and a mixed probe solution 2 of chromogenic probe C and D with a concentration of 0.5 μM each for standby.

[0038] S4: Drop the freshly prepared mixed probe solution 1 onto the chip substrate loaded with capture probe A after S1 cleaning, and drop the freshly prepared mixed probe solution 2 onto the chip substrate loaded with capture probe B after cleaning. React at 25 °C for 30 min respectively. After gently washing 3 times with 1×PBST buffer solution, place them under a fluorescence microscope and record the fluorescence microscopic images of Cy5 and FAM respectively.

[0039] As Figure 2 shown, for the mutant target, the beads on the chip loaded with capture probe A exhibit obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B do not emit light in the FAM fluorescence channel; for the wild-type target, the beads on the chip loaded with capture probe A do not emit light in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B exhibit obvious fluorescence in the FAM fluorescence channel; for the heterozygous (mutant + wild) target, the beads on the chip loaded with capture probe A exhibit obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B also exhibit obvious fluorescence in the FAM fluorescence channel; the experimental results are the same as the expected results, indicating that this chip can be used for SNP1 genotyping detection and analysis.

[0040] Example 3. Detect SNP2.

[0041] I. Target sequences and probes, as shown in Table 2.

[0042] Table 2

[0043] II. Target sequence comparison test.

[0044] The differences from Example 2 are as follows: 1. The concentrations of hairpin probes A and B are 0.4 μM. Execute the temperature program: heat at 90 °C for 10 min and maintain at 4 °C for 20 min to obtain the reacted mixed solution.

[0045] 2. The reacted mixed solution hybridizes on the chip substrate at 20 °C for 40 min.

[0046] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 1 μM.

[0047] 4. Drop mixed probe solution 1 or mixed probe solution 2 on the chip substrate and react at 30 °C for 25 min.

[0048] As Figure 3As shown in the figure, for the mutant target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, while the beads on the chip loaded with capture probe B do not emit light in the FAM fluorescence channel; for the wild-type target, the beads on the chip loaded with capture probe A do not emit light in the Cy5 fluorescence channel, while the beads on the chip loaded with capture probe B show obvious fluorescence in the FAM fluorescence channel; for the heterozygous (mutant + wild) target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B also show obvious fluorescence in the FAM fluorescence channel; the experimental results are the same as the expected results, indicating that this chip can be used for SNP2 genotyping detection and analysis.

[0049] Example 4. Detect SNP3.

[0050] I. Target sequence and probes, as shown in Table 3.

[0051] Table 3

[0052] II. Target sequence comparison test.

[0053] The differences from Example 2 are as follows: 1. The concentrations of hairpin probes A and B are 0.8 μM. Execute the temperature program: heat at 85 °C for 10 min and keep at 8 °C for 15 min to obtain the reaction mixture.

[0054] 2. Hybridize the reaction mixture on the chip substrate at 35 °C for 20 min.

[0055] 3. The concentration of mixed probe solution 1 or mixed probe solution 2 is 1.5 μM.

[0056] 4. Drop mixed probe solution 1 or mixed probe solution 2 on the chip substrate and react at 20 °C for 10 min.

[0057] As Figure 4 shown, for the mutant target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, while the beads on the chip loaded with capture probe B do not emit light in the FAM fluorescence channel; for the wild-type target, the beads on the chip loaded with capture probe A do not emit light in the Cy5 fluorescence channel, while the beads on the chip loaded with capture probe B show obvious fluorescence in the FAM fluorescence channel; for the heterozygous (mutant + wild) target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B also show obvious fluorescence in the FAM fluorescence channel; the experimental results are the same as the expected results, indicating that this chip can be used for SNP3 genotyping detection and analysis.

[0058] Example 5. Detection of SNP4.

[0059] I. Target sequence and probes are shown in Table 4.

[0060] Table 4

[0061] II. Comparative test of target sequences.

[0062] The differences from Example 2 are as follows: 1. The concentrations of hairpin probes A and B are 0.2 μM. Execute the temperature program: heat at 95 °C for 10 min and hold at 0 °C for 10 min.

[0063] 2. The hybridized reaction of the mixed solution after reaction on the chip substrate is carried out at 20 °C for 20 min.

[0064] 3. The concentrations of both mixed probe solution 1 and mixed probe solution 2 are 2 μM.

[0065] 4. Drop mixed probe solution 1 or mixed probe solution 2 on the chip substrate and react at 30 °C for 60 min.

[0066] As Figure 5 shown, for the mutant target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B do not emit light in the FAM fluorescence channel; for the wild-type target, the beads on the chip loaded with capture probe A do not emit light in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B show obvious fluorescence in the FAM fluorescence channel; for the heterozygous (mutant + wild) target, the beads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the beads on the chip loaded with capture probe B also show obvious fluorescence in the FAM fluorescence channel; the experimental results are the same as the expected results, indicating that this chip can be used for SNP4 genotyping detection and analysis.

[0067] Example 6. Detection of SNP5.

[0068] I. Target sequence and probes are shown in Table 5.

[0069] Table 5

[0070] II. Comparative test of target sequences.

[0071] The differences from Example 2 are as follows: 1. The concentrations of hairpin probes A and B are 1.0 μM. Execute the temperature program: heat at 90 °C for 5 min and hold at 10 °C for 20 min to obtain the mixed solution after the reaction.

[0072] 2. Hybridize the mixed solution after the reaction on the chip substrate at 40 °C for 20 min.

[0073] 3. The concentrations of both mixed probe solution 1 and mixed probe solution 2 are 0.8 μM.

[0074] 4. Drop mixed probe solution 1 or mixed probe solution 2 on the chip substrate and react at 40 °C for 30 min.

[0075] As Figure 6 shown, for the mutant target, the microbeads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the microbeads on the chip loaded with capture probe B do not emit light in the FAM fluorescence channel; for the wild-type target, the microbeads on the chip loaded with capture probe A do not emit light in the Cy5 fluorescence channel, and the microbeads on the chip loaded with capture probe B show obvious fluorescence in the FAM fluorescence channel; for the heterozygous (mutant + wild) target, the microbeads on the chip loaded with capture probe A show obvious fluorescence in the Cy5 fluorescence channel, and the microbeads on the chip loaded with capture probe B also show obvious fluorescence in the FAM fluorescence channel; the experimental results are the same as the expected results, indicating that this chip can be used for SNP5 genotyping detection and analysis.

[0076] Example 7. Erasure and reuse test 1 of the chip.

[0077] 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.

[0078] The mutant target sequence test is the same as that in Example 2.

[0079] Alkaline washing of the microbead solid-phase chip substrate: Drop the used chip with an alkaline solution with a pH of 12 and incubate and wash at room temperature 3 times, 10 min each time, and wash with 1×PBST until neutral.

[0080] Repeat the above mutant target sequence test and alkaline washing steps of the microbead solid-phase chip substrate 10 times.

[0081] The results are as Figure 7 shown. After each use of the gene chip, effective erasure can be achieved by washing with an alkaline solution, the hybridization probes are removed, and the fluorescence disappears. It functions normally when used again, can accurately identify the mutant target sequence, and has good reuse performance.

[0082] Example 8. Erasure and reuse test 2 of the chip.

[0083] The target sequence, 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.

[0084] The test of the wild-type target sequence is the same as that in Example 2.

[0085] For the alkali washing of the microbead solid-phase chip substrate to erase the used chip, an alkaline solution with a pH of 12 was added dropwise, incubated at room temperature for 3 times, 10 min each time, and washed with 1×PBST until neutral.

[0086] Repeat the above steps of the wild-type target sequence test and the alkali washing of the microbead solid-phase chip substrate 10 times.

[0087] The results are as Figure 8 shown. After each use of the gene chip, effective erasure can be achieved by washing with an alkaline solution. The hybridization probes are removed and the fluorescence disappears. It functions normally when used again, can accurately identify the wild-type target sequence, and has good reuse performance.

[0088] Comparative example. Comparison between this application and the erasable chip technology based on ligase.

[0089] The mutant target sequence and the wild-type target sequence are the same as those in Example 2. The target sequences were respectively compared and tested by the microbead chip technology of the current invention and the erasable chip technology based on ligase (CN 119193793 A).

[0090] The test method and probe design of the microbead chip technology of the current invention are the same as those in Example 2.

[0091] The test method and probe design of the erasable chip technology based on ligase are shown in Table 6. The chip preparation and detection scheme refer to CN 119193793 A: Table 6

[0092] The results of the comparative test are as Figure 9 shown. To distinguish the same mutant target or wild-type target, the detection specificity of the technology of the present invention is significantly better, specifically manifested as lower false positive signals and higher discrimination factors (discrimination factor = true signal / false positive signal), Figure 9 and the specific values are shown in Table 7: Table 7

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, colorimetric probe A, colorimetric probe B, colorimetric probe C and colorimetric probe D; the capture probes include capture probe A and capture probe B.

2. The chip according to claim 1, wherein The sequence of the hairpin probe A sequentially includes a stem sequence 1 of 10-16 bases, a loop sequence 1 of 10-16 bases, a stem sequence 2 of 10-16 bases and a strand hybridization trigger sequence 1 of 16-30 bases from the 5'-end to the 3'-end. The stem sequence 1 and the loop sequence 1 are completely complementary to the wild-type target sequence and form a mismatch at the mutation site with the mutant target sequence, and the mismatch site is located in the stem sequence 1.

3. The chip according to claim 1, wherein The sequence of the hairpin probe B sequentially includes a strand hybridization trigger sequence 2 of 16-30 bases, a stem sequence 3 of 10-16 bases, a loop sequence 2 of 10-16 bases and a stem sequence 4 of 10-16 bases from the 5'-end to the 3'-end. The loop sequence 2 and the stem sequence 4 are completely complementary to the mutant target and form a mismatch at the mutation site with the wild-type target sequence, and the mismatch site is located in the stem sequence 4.

4. The chip according to claim 1, wherein The capture probe A sequentially includes a transition sequence 1 of 0-15 bases and a capture sequence 1 of 10-16 bases from the 3'-end to the 5'-end, and the capture sequence 1 is completely or partially the same as the loop sequence 1 of the hairpin probe A.

5. The chip according to claim 1, wherein The capture probe B sequentially includes a transition sequence 2 of 0-15 bases and a capture sequence 2 of 10-16 bases from the 5'-end to the 3'-end, and the capture sequence 2 is completely or partially the same as the loop sequence 2 of the hairpin probe B.

6. The chip according to claim 1, wherein 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 are both 20-60 bases.

7. The chip according to claim 1, wherein 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 are both 20-60 bases.

8. A detection method using the reusable genotyping chip according to any one of claims 1-7, characterized in that: The specific steps of this detection method are as follows: S1: Prepare a reaction solution and heat it. The reaction solution includes hairpin probe A, hairpin probe B and the target DNA of the sample to be tested. S2: Drop the reaction solution onto the chip substrate loaded with capture probe A or capture probe B for hybridization reaction, and after the reaction, wash it with a buffer solution. S3: Prepare a mixed probe solution 1 from colorimetric probe A and B, and drop it onto the chip substrate loaded with capture probe A after washing for reaction; prepare a mixed probe solution 2 from colorimetric probe C and D, and drop it onto the chip substrate loaded with capture probe B after washing for reaction, then wash it with a buffer solution and place it under a fluorescence microscope for photographing and recording. Single nucleotide polymorphism genotyping detection can be achieved according to the color of the fluorescence image. S4: Drop an alkaline solution onto the used chip for incubation and washing to erase the colorimetric probe. The chip after erasure can be used again according to the standard detection process.

9. The detection method according to claim 8, wherein The pH of the alkaline solution in S4 is 12.

10. The detection method according to claim 8, wherein The concentration of the hairpin probe A or B in S1 is 0.2-1 μM. The heating is first at 85-95 °C for 5-10 min, and then maintained at 0-10 °C for 10-20 min.

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