An erasable single nucleotide polymorphism genotyping chip and detection method

Abstract

This invention belongs to the field of biochip technology, specifically an erasable single nucleotide polymorphism (SNP) genotyping chip and its detection method. The erasable SNP genotyping chip provided by this invention is prepared using polystyrene microspheres, capture probes, and a silicon substrate. In the SNP recognition stage, it relies on a FEN1 enzyme-mediated invasive reaction. Based on the technical characteristics of this invasive reaction, downstream probes A and B are designed with self-protective hairpin structures, preventing false positive signals from hybridization with the capture probe even when added in excess, thus significantly improving detection specificity. The detection method of the erasable SNP genotyping chip provided by this invention overcomes the long-standing problem of high application costs caused by the single-use nature of solid-phase SNP gene detection chips.

Description

Technical Field

[0001] This application relates to the field of biochip technology, and in particular to an erasable single nucleotide polymorphism genotyping chip and its detection technology. Background Technology

[0002] Biochip technology is a cutting-edge biotechnology that emerged in the 1980s. This technology uses materials such as glass, silicon wafers, and polymers as substrates to integrate and immobilize various biomolecular probes at high density, enabling high-throughput analysis of biological samples using the principle of specific molecular recognition. Based on the type of immobilized probes, biochips can be mainly divided into three categories: gene chips, protein chips, and tissue chips. Gene chips typically use solid-phase carriers such as microbeads, on which specific oligonucleotide sequences or gene fragments are immobilized as molecular probes. Through the principle of complementary base pairing, these probes can specifically hybridize with the target nucleic acid sequence. By detecting the hybridization signal using a fluorescence imaging system, optical information can be converted into biological data. This technology has significant application value in fields such as gene expression profiling, mutation screening, and genomic polymorphism research.

[0003] Single nucleotide polymorphisms (SNPs) are genetic polymorphisms caused by variations in a single base in the genome. As important genetic markers, they play a crucial role in species genetic research. SNP sites are numerous and widely distributed. Due to the high throughput and convenient detection advantages of gene chips, chip technology has shown great potential in the field of SNP detection. However, traditional solid-phase SNP detection chips, which use single-base extension or DNA ligation techniques for mutation identification, suffer from the technical limitation of non-reusability, resulting in high detection costs and limiting their large-scale application. Therefore, the development of erasable single nucleotide polymorphism genotyping chips and their detection technologies is of significant scientific and practical value for promoting the widespread application of gene chips in SNP detection. Summary of the Invention

[0004] This invention provides an erasable single nucleotide polymorphism (SNP) genotyping chip. This gene chip detection technology solves the problem that existing solid-phase SNP gene detection chips can only be used once, resulting in high application costs.

[0005] An erasable single nucleotide polymorphism (SNP) genotyping chip is prepared using polystyrene microspheres, a capture probe, and a silicon substrate. The primers used with the chip include an upstream probe, a downstream probe A, a downstream probe B, a chromogenic probe A, a chromogenic probe B, a chromogenic probe C, and a chromogenic probe D.

[0006] Preferably, the upstream probe is 12 to 25 bases in length, and the sequence of the upstream probe from the 5' end to the 3' end is continuously complementary to the target sequence up to the base before the mutation site, forming a base mismatch at the mutation site.

[0007] Preferably, the sequence of the downstream probe A, from the 3' end to the 5' end, includes a toe sequence of 10-20 bases, a stem sequence 1 of 10-20 bases, a loop sequence 1 of 3-10 bases, a stem sequence 2 of 10-20 bases, and a strand hybridization trigger sequence 1 of 16-30 bases; the stem sequence 1 and the stem sequence 2 are self-complementary; the downstream probe A, from the 3' end to the 5' end, is continuously complementary to the mutant target sequence to the mutation site.

[0008] Preferably, the downstream probe B sequence comprises, from the 3' end to the 5' end, a toe sequence of 10-20 bases, a stem sequence 3 of 10-20 bases, a loop sequence 2 of 3-10 bases, a stem sequence 4 of 10-20 bases, and a strand hybridization trigger sequence 2 of 16-30 bases; the stem sequence 3 and the stem sequence 4 are self-complementary; and the downstream probe B is continuously complementary to the wild-type target sequence from the 3' end to the 5' end to the mutation site.

[0009] Preferably, the sequence of the capture probe includes a transition sequence of 0 to 15 bases and a capture sequence of 10 to 20 bases from the 5' end to the 3' end, and the capture sequence is completely or partially identical to the stem sequence 1 of the downstream probe A.

[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 length of each is 20 to 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 length of each is 20 to 60 bases.

[0012] A detection method using an erasable single nucleotide polymorphism (SNP) genotyping chip, characterized by the following steps:

[0013] S1: Prepare and incubate the reaction solution, which includes FEN1 enzyme, upstream probe, downstream probe A, downstream probe B and target DNA of the sample to be tested;

[0014] S2: Add the reaction solution to the erasable single nucleotide polymorphism genotyping chip for hybridization reaction, and wash with buffer solution after the reaction is completed;

[0015] S3: Add a chromogenic probe mixture to the cleaned chip, then clean it with buffer and place it under a fluorescence microscope to take pictures and record the data. The single nucleotide polymorphism gene typing can be achieved based on the color of the fluorescence image.

[0016] S4: After use, the chip can be incubated and cleaned with an alkaline solution to remove the colorimetric probe. The chip can then be reused according to the standard testing procedure.

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

[0018] Preferably, the upstream probe concentration in S1 is 10~100 nM, the concentrations of downstream probe A and downstream probe B are both 100~1000 nM, the FEN1 enzyme is 2~10 U, the incubation temperature is 55~65 ℃, and the incubation time is 0.5 h~3 h.

[0019] Beneficial effects

[0020] This invention provides an erasable single nucleotide polymorphism (SNP) genotyping chip and detection method. This chip detection technology overcomes the long-standing problem that solid-phase SNP gene detection chips can only be used once, resulting in high application costs. It realizes the erasable reuse of SNP gene detection chips, which can significantly reduce the application cost of solid-phase gene chips when used for large-scale sample genotyping.

[0021] The erasable single nucleotide polymorphism (SNP) genotyping chip provided by this invention significantly improves detection specificity compared to existing ligase-based erasable chips. Ligase-based erasable chips require excessive addition of straight-chain primers (probes A, B, and C) during the SNP recognition stage. To prevent false positives, the capture probes linked to the beads need to be pre-conjugated with blocking probes. However, the inherent thermodynamic equilibrium of DNA hybridization prevents the blocking probes from achieving perfect full-blocking; excessive primers can still hybridize with the capture probes, leading to false positives. In contrast, the erasable chip of this invention relies on a FEN1 enzyme-mediated invasive reaction during the SNP recognition stage. Based on the characteristics of this invasive reaction, downstream probes A and B are designed with self-protective hairpin structures. Excess primers will not hybridize with the capture probes, preventing false positives and significantly improving detection specificity. Furthermore, the hairpin structures of downstream probes A and B have a self-protective function, allowing the capture probes on the beads to be used directly without blocking, simplifying the detection and chip activation process. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the technical principle of the erasable single nucleotide polymorphism genotyping chip of this application;

[0023] Figure 2 The image shown is a fluorescence micrograph of the SNP1 genotyping detection in Example 2 of this application.

[0024] Figure 3 The image shown is a fluorescence micrograph of the SNP2 genotyping detection in Example 3 of this application.

[0025] Figure 4 The image shown is a fluorescence micrograph of the SNP3 genotyping detection in Example 4 of this application.

[0026] Figure 5 The image shown is a fluorescence micrograph of the SNP4 genotyping detection in Example 5 of this application.

[0027] Figure 6 The image shown is a fluorescence micrograph of the SNP5 genotyping detection in Example 6 of this application.

[0028] Figure 7 Fluorescence micrographs of the gene chip erasure and reuse test in Example 7 of this application;

[0029] Figure 8 Fluorescence micrographs of the gene chip erasure and reuse test in Example 8 of this application;

[0030] Figure 9 This is a comparative image showing the two gene chip technologies. Detailed Implementation

[0031] Reagents used in the examples:

[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] FEN1 enzyme reaction buffer (final concentration): 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton®X-100.

[0034] Example 1. Preparation of an erasable single nucleotide polymorphism genotyping chip.

[0035] A schematic diagram illustrating the principle of erasable single nucleotide polymorphism (SNP) genotyping chip technology, as shown below. Figure 1 .

[0036] 1. Solid-phase chip substrate with capture probe mounted.

[0037] 1. Polystyrene microsphere activation steps: Prepare a suspension of 5 mg / mL of carboxylated polystyrene microspheres with pre-prepared morpholine ethanesulfonic acid buffer (MES, 0.1M, pH 6); then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and N-hydroxysuccinimide (NHS) solution prepared using MES, so that the concentration of EDC and NHS in the suspension system reaches 25 mg / mL, shake for 1 h, and obtain the activated polystyrene microsphere suspension.

[0038] 2. Polystyrene microsphere covalent binding of capture probe: Dissolve 10 nmol of the capture probe powder to be ligated in MES buffer, then mix with the activated polystyrene microsphere suspension to a polystyrene microsphere concentration of 5 mg. Incubate at room temperature with shaking for 4 hours. Then, wash three times with PBST buffer (5000 rpm, 5 min), and resuspend in ultrapure water to obtain a solution of polystyrene microspheres covalently bound to oligonucleotide chains.

[0039] 3. A groove array region is formed on the first surface of a silicon substrate (10 mm × 10 mm × 1 mm) using photolithography.

[0040] 4. The polystyrene covalently bonded oligonucleotide microbeads 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 into the mounting groove (10 mm × 10 mm × 2 mm) in the assembly mold (75 mm × 25 mm × 3 mm). 150 μL of monodisperse microbead solution was uniformly dropped onto the first surface of the silicon substrate. A glass slide was placed on the assembly mold, and after ensuring that there was no air in the liquid under the glass slide, the mold was sealed with sealing film. The substrate was then placed face up in an ultrasonic cleaner and ultrasonically assembled for 10 min at 120 W. The surface of the silicon substrate was cleaned twice with deionized water to complete the assembly of the microbeads and prepare a solid-phase chip substrate loaded with a capture probe.

[0041] II. Probe preparation method.

[0042] Upstream probe: 12-25 bases in length, continuously complementary to the target sequence from the 5' end to the 3' end up to the base before the mutation site, forming a base mismatch at the mutation site;

[0043] Downstream probe A: From the 3' end to the 5' end, it includes a toe sequence of 10-20 bases, a stem sequence 1 of 10-20 bases, a loop sequence 1 of 3-10 bases, a stem sequence 2 of 10-20 bases, and a strand hybridization trigger sequence 1 of 16-30 bases. Downstream probe A is continuously complementary to the mutant target sequence to the mutation site from the 3' end to the 5' end. The stem sequence 1 and stem sequence 2 of downstream probe A are self-complementary.

[0044] Downstream probe B: From the 3' end to the 5' end, it includes a toe sequence of 10-20 bases, a stem sequence 3 of 10-20 bases, a loop sequence 2 of 3-10 bases, a stem sequence 4 of 10-20 bases, and a strand hybridization trigger sequence 2 of 16-30 bases. Downstream probe B is continuously complementary to the wild-type target sequence to the mutation site from the 3' end to the 5' end. The stem sequence 3 and stem sequence 4 of downstream probe B are self-complementary.

[0045] The capture probe consists of a transition sequence of 0 to 15 bases and a capture sequence of 10 to 20 bases, which are identical or partially identical to the stem sequence 1 of the downstream probe A.

[0046] Fluorescent chromogenic probes: The 5' ends of fluorescent chromogenic probes A and B are modified with the same fluorescent chromogenic group, and the 5' ends of fluorescent chromogenic probes C and D are modified with the same fluorescent chromogenic group. The sequence length of all fluorescent chromogenic probes is 20-60 bases, and all can fold to form hairpin structures. The chain hybridization triggering sequence 1 of downstream probe A can trigger the alternating hairpin opening self-assembly of fluorescent chromogenic probes A and B, and the chain hybridization triggering sequence 2 of downstream probe B can trigger the alternating hairpin opening self-assembly of fluorescent chromogenic probes C and D.

[0047] Example 2. Detection of SNP1.

[0048] I. Target sequence and probe, as shown in Table 1.

[0049] Table 1

[0050]

[0051] II. Target sequence comparison test.

[0052] S1: Prepare 100 μL of reaction solution using the reaction buffer of FEN1 enzyme, which contains (final concentration): 5 U FEN1, 25 nM upstream probe, 250 nM downstream probe A, 250 nM downstream probe B, and 25 nM target DNA (target sequence mutant or target sequence wild type). Incubate at 65°C for 1 h to obtain the mixed solution after reaction.

[0053] S2: The mixed solution after the reaction was dropped onto the solid-phase chip substrate loaded with the capture probe as described in Example 1, and the hybridization reaction was carried out at 25°C for 30 min. The mixture was then gently washed three times with 1×PBST buffer.

[0054] S3: Prepare chromogenic probe A, B, C, and D solutions with a concentration of 2 μM using 1×PBST. Heat each solution at 95 °C for 5 min and allow them to cool naturally to room temperature. Then mix the four probe solutions to obtain a 0.5 μM fluorescent chromogenic probe mixed solution.

[0055] S4: Add freshly prepared fluorescent chromogenic probe mixture solution to the chip substrate cleaned in S2, react at 25 ℃ for 30 min, gently wash three times with 1×PBST buffer solution, and then place under a fluorescence microscope to record the fluorescence microscopic images of FAM and ROX respectively.

[0056] like Figure 2 As shown, the microbeads on the wild-type target chip exhibit obvious FAM green fluorescence, while the microbeads in the ROX yellow fluorescence channel do not emit light; the microbeads on the mutant target chip exhibit obvious ROX yellow fluorescence, while the microbeads in the FAM green fluorescence channel do not emit light; the microbeads on the heterozygous (mutant + wild) target chip emit light in both the FAM green fluorescence channel and the ROX yellow fluorescence channel; the experimental results are the same as expected, indicating that this chip can be used for SNP1 genotyping detection and analysis.

[0057] Example 3. Detection of SNP2.

[0058] I. Target sequence and probe, as shown in Table 2.

[0059] Table 2

[0060]

[0061] II. Target sequence comparison test.

[0062] The difference from Example 2 is as follows:

[0063] 1. The reaction solution is: 2 U FEN 1, 50 nM upstream probe, 500 nM downstream probe A, 500 nM downstream probe B, and 25 nM target DNA (target sequence mutant or target sequence wild type). Incubate at 60°C for 2 h to obtain the mixed solution after the reaction.

[0064] 2. The mixed solution after the reaction was dropped onto the solid-phase chip substrate loaded with the capture probe, and the hybridization reaction was carried out at 20 °C for 40 min.

[0065] 3. The concentration of the fluorescent chromogenic probe mixed solution is 1 μM.

[0066] 4. The fluorescent chromogenic probe mixture was reacted on the chip substrate at 30 °C for 25 min.

[0067] like Figure 3As shown, the microbeads on the wild-type target chip exhibit obvious FAM green fluorescence, while the microbeads in the ROX yellow fluorescence channel do not emit light; the microbeads on the mutant target chip exhibit obvious ROX yellow fluorescence, while the microbeads in the FAM green fluorescence channel do not emit light; the microbeads on the heterozygous (mutant + wild) target chip emit light in both the FAM green fluorescence channel and the ROX yellow fluorescence channel; the experimental results are consistent with the expected results, indicating that this chip can be used for SNP2 genotyping detection and analysis.

[0068] Example 4. Detection of SNP3.

[0069] I. Target sequence and probe, as shown in Table 3.

[0070] Table 3

[0071]

[0072] II. Target sequence comparison test.

[0073] The difference from Example 2 is as follows:

[0074] 1. The reaction solution is: 6 U FEN 1, 10 nM upstream probe, 200 nM downstream probe A, 200 nM downstream probe B, and 25 nM target DNA (target sequence mutant or target sequence wild type). Incubate at 65°C for 3 h to obtain the mixed solution after the reaction.

[0075] 2. The mixed solution after the reaction was dropped onto the solid-phase chip substrate loaded with the capture probe, and the hybridization reaction was carried out at 35 °C for 20 min.

[0076] 3. The concentration of the fluorescent chromogenic probe mixture solution is 1.5 μM.

[0077] 4. The fluorescent chromogenic probe mixture was reacted on the chip substrate at 20 °C for 10 min.

[0078] like Figure 4 As shown, the microbeads on the wild-type target chip exhibit obvious FAM green fluorescence, while the microbeads in the ROX yellow fluorescence channel do not emit light; the microbeads on the mutant target chip exhibit obvious ROX yellow fluorescence, while the microbeads in the FAM green fluorescence channel do not emit light; the microbeads on the heterozygous (mutant + wild) target chip emit light in both the FAM green fluorescence channel and the ROX yellow fluorescence channel; the experimental results are consistent with the expected results, indicating that this chip can be used for SNP3 genotyping detection and analysis.

[0079] Example 5. Detection of SNP4.

[0080] I. Target sequence and probe, as shown in Table 4.

[0081] Table 4

[0082]

[0083] II. Target sequence comparison test.

[0084] The difference from Example 2 is as follows:

[0085] 1. The reaction solution is: 8 U FEN 1, 50 nM upstream probe, 400 nM downstream probe A, 400 nM downstream probe B, and 25 nM target DNA (target sequence mutant or target sequence wild type). Incubate at 55°C for 1.5 h to obtain the mixed solution after the reaction.

[0086] 2. The mixed solution after the reaction was dropped onto the solid-phase chip substrate loaded with the capture probe, and the hybridization reaction was carried out at 20 °C for 20 min.

[0087] 3. The concentration of the fluorescent chromogenic probe mixture solution is 2 μM.

[0088] 4. The fluorescent chromogenic probe mixture was reacted on the chip substrate at 30 °C for 60 min.

[0089] like Figure 5 As shown, the microbeads on the wild-type target chip exhibit obvious FAM green fluorescence, while the microbeads in the ROX yellow fluorescence channel do not emit light; the microbeads on the mutant target chip exhibit obvious ROX yellow fluorescence, while the microbeads in the FAM green fluorescence channel do not emit light; the microbeads on the heterozygous (mutant + wild) target chip emit light in both the FAM green fluorescence channel and the ROX yellow fluorescence channel; the experimental results are consistent with the expected results, indicating that this chip can be used for SNP4 genotyping detection and analysis.

[0090] Example 6. Detection of SNP5.

[0091] I. Target sequence and probe, as shown in Table 5.

[0092] Table 5

[0093]

[0094] II. Target sequence comparison test.

[0095] The difference from Example 2 is as follows:

[0096] 1. The reaction solution is: 10 U FEN 1, 100 nM upstream probe, 1000 nM downstream probe A, 1000 nM downstream probe B, and 25 nM target DNA (target sequence mutant or target sequence wild type). Incubate at 60°C for 0.5 h to obtain the mixed solution after the reaction.

[0097] 2. The mixed solution after the reaction was dropped onto the solid-phase chip substrate loaded with the capture probe, and the hybridization reaction was carried out at 40 °C for 20 min.

[0098] 3. The concentration of the fluorescent chromogenic probe mixture solution is 0.8 μM.

[0099] 4. The fluorescent chromogenic probe mixture was reacted on the chip substrate at 40 °C for 30 min.

[0100] like Figure 6 As shown, the microbeads on the wild-type target chip exhibit obvious FAM green fluorescence, while the microbeads in the ROX yellow fluorescence channel do not emit light; the microbeads on the mutant target chip exhibit obvious ROX yellow fluorescence, while the microbeads in the FAM green fluorescence channel do not emit light; the microbeads on the heterozygous (mutant + wild) target chip emit light in both the FAM green fluorescence channel and the ROX yellow fluorescence channel; the experimental results are consistent with the expected results, indicating that this chip can be used for SNP5 genotyping detection and analysis.

[0101] Example 7. Chip erasure and reuse test 1.

[0102] 1. The target sequence mutant, upstream probe, downstream probe A, downstream probe B, capture probe, chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D are the same as in Example 2.

[0103] 2. The mutant target sequence test is the same as in Example 2.

[0104] 3. Alkali washing and wiping of the microbead solid-phase chip substrate.

[0105] After use, the chip was incubated with an alkaline solution at pH 12 at room temperature for 3 times, 10 minutes each time, and then washed with 1×PBST until neutral.

[0106] 4. Repeat the target sequence comparison test and the alkaline washing and erasure steps of the microbead solid-phase chip substrate 10 times. The results are as follows: Figure 7 As shown, the gene chip can be effectively erased by washing with an alkaline solution after each use; the chromogenic probe is removed and the fluorescence disappears. It functions normally upon reuse, accurately identifying mutant target sequences, and exhibits good reusability.

[0107] Example 8. Chip erasure and reuse test 2.

[0108] 1. The target sequence wild type, upstream probe, downstream probe A, downstream probe B, capture probe, chromogenic probe A, chromogenic probe B, chromogenic probe C, and chromogenic probe D are the same as in Example 2.

[0109] 2. Wild-type target sequence testing is the same as in Example 2.

[0110] 3. Alkali washing and wiping of the microbead solid-phase chip substrate.

[0111] After use, the chip was incubated with an alkaline solution at pH 12 at room temperature for 3 times, 10 minutes each time, and then washed with 1×PBST until neutral.

[0112] 4. Repeat the target sequence test and alkaline washing / erasing steps for the microbead solid-phase chip substrate 10 times. Figure 8 As shown, the gene chip can be effectively erased by washing with an alkaline solution after each use, removing the chromogenic probe and eliminating fluorescence. It functions normally upon reuse, accurately identifying wild-type target sequences, and exhibits good reusability.

[0113] Comparative example. A comparison of the above-mentioned chip with erasable chip technology based on ligase.

[0114] The target sequence mutant and target sequence wild type were the same as in Example 2, and the target sequence was compared and tested using the microbead chip technology and ligase-based erasable chip technology (CN 119193793 A) of the present invention.

[0115] The microbead chip technology testing method and probe design of the present invention are the same as in Example 2.

[0116] Table 6 shows the testing methods and probe design for erasable chip technology based on ligases. The chip fabrication and detection scheme refer to CN 119193793 A.

[0117] Table 6

[0118]

[0119] Comparison test results as follows Figure 9 As shown, when distinguishing between the same mutant or wild-type targets, the chip of this invention has better detection specificity, specifically manifested in lower false positive signals and a higher discriminant factor (discriminant factor = true signal / false positive signal). Figure 9 The specific values ​​are shown in Table 7:

[0120] Table 7

[0121]

Claims

1. An erasable single nucleotide polymorphism (SNP) genotyping chip, characterized in that, The chip is prepared by polystyrene microspheres, a capture probe and a silicon substrate. The primers used with the chip include an upstream probe, a downstream probe A, a downstream probe B, a colorimetric probe A, a colorimetric probe B, a colorimetric probe C and a colorimetric probe D. The upstream probe is 12 to 25 bases in length. The sequence of the upstream probe from the 5' end to the 3' end is continuously complementary to the target sequence up to the base before the mutation site, forming a base mismatch at the mutation site. The downstream probe A sequence, from the 3' end to the 5' end, includes a toe sequence of 10-20 bases, a stem sequence 1 of 10-20 bases, a loop sequence 1 of 3-10 bases, a stem sequence 2 of 10-20 bases, and a strand hybridization trigger sequence 1 of 16-30 bases; the stem sequence 1 and the stem sequence 2 are self-complementary; the downstream probe A is continuously complementary to the mutant target sequence from the 3' end to the 5' end to the mutation site; The downstream probe B sequence, from the 3' end to the 5' end, includes a toe sequence of 10-20 bases, a stem sequence 3 of 10-20 bases, a loop sequence 2 of 3-10 bases, a stem sequence 4 of 10-20 bases, and a strand hybridization trigger sequence 2 of 16-30 bases; the stem sequence 3 and the stem sequence 4 are self-complementary; the downstream probe B is continuously complementary to the wild-type target sequence from the 3' end to the 5' end to the mutation site; The sequence of the capture probe includes a transition sequence of 0 to 15 bases and a capture sequence of 10 to 20 bases from the 5' end to the 3' end. The capture sequence is completely or partially the same as the stem sequence 1 of the downstream probe A. 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 length of each is 20 to 60 bases. The 5' ends of the fluorescent chromogenic probes C and D are modified with the same fluorescent chromogenic group, and the sequence length of each is 20 to 60 bases.

2. A method for detecting erasable single nucleotide polymorphism genotyping chips according to claim 1 for non-diagnostic and non-therapeutic purposes, characterized in that, The steps of this detection method are as follows: S1: Prepare and incubate the reaction solution, which includes FEN1 enzyme, upstream probe, downstream probe A, downstream probe B and target DNA of the sample to be tested; S2: Add the reaction solution to the erasable single nucleotide polymorphism genotyping chip for hybridization reaction, and wash with buffer solution after the reaction is completed; S3: Add a chromogenic probe mixture to the cleaned chip, then clean it with buffer and place it under a fluorescence microscope to take pictures and record the data. The single nucleotide polymorphism gene typing can be achieved based on the color of the fluorescence image. S4: After use, the chip can be incubated and cleaned with an alkaline solution to remove the colorimetric probe. The chip can then be reused according to the standard testing procedure.

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

4. The detection method according to claim 2, characterized in that, The upstream probe concentration in S1 is 10~100 nM, the downstream probe A and downstream probe B are both 100~1000 nM, the FEN1 enzyme is 2~10 U, the incubation temperature is 55~65 ℃, and the incubation time is 0.5 h~3 h.

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