CN118562930A

CN118562930A - SNP detection method based on liquid chip

Info

Publication number: CN118562930A
Application number: CN202310985818.2A
Authority: CN
Inventors: 李雅雅; 沙海天; 白艳军
Original assignee: Shanghai Wanzijian Biotechnology Co ltd
Current assignee: Shanghai Wanzijian Biotechnology Co ltd
Priority date: 2023-08-07
Filing date: 2023-08-07
Publication date: 2024-08-30

Abstract

The application relates to the technical field of biology, in particular to a SNP detection method based on a liquid chip. The application provides a SNP detection primer based on a liquid chip, which comprises an upstream primer and a downstream primer; the upstream primer sequentially comprises a tag sequence, a partition wall modification, a first specific sequence and a first enzyme cutting sequence from a 5 'end to a 3' end; the downstream primer sequentially comprises a detection group, a second specific sequence and a second enzyme cutting sequence from the 5 'end to the 3' end, wherein the first enzyme cutting sequence or the second enzyme cutting sequence contains RNA bases matched with SNP loci of target genes. According to the application, the enzyme cutting sequence is designed on the primer, and the RNA base in the primer can be cut by RNase enzyme only after the upstream primer, the downstream primer and the target gene are completely matched, so that normal extension is realized, thereby reducing primer dimer and other non-specific amplification products, and realizing single-tube multiplex amplification of PCR; multiple targets can be detected through the quantum dot microsphere, and simultaneous detection of multiple SNP loci can be realized.

Description

SNP detection method based on liquid chip

Technical Field

The application relates to the technical field of biology, in particular to a SNP detection method based on a liquid chip.

Background

Polymerase Chain Reaction (PCR) is a molecular biological technique for amplifying specific DNA fragments, and the greatest feature of PCR is the ability to greatly increase minute amounts of DNA. The basic principle of PCR consists of three basic reaction steps of denaturation-annealing-extension: (1) denaturation of template DNA: heating the template DNA to 93 ℃ for a certain time, and then dissociating the double-stranded template DNA or the double-stranded DNA formed by PCR amplification to make the template DNA become single-stranded so that the template DNA is combined with the primer to prepare for the next round of reaction; (2) annealing (renaturation) of template DNA to primer: after the template DNA is denatured into single strands by heating, the temperature is reduced to about 55 ℃, and the primer is combined with the complementary sequence of the single strands of the template DNA in a pairing way; (3) primer extension: under the action of DNA polymerase (such as Taq DNA polymerase) at 72 deg.C, the DNA template-primer conjugate uses dNTP as reaction raw material and target sequence as template, and synthesizes a new semi-reserved copy chain complementary with template DNA chain according to the base complementary pairing and semi-reserved copy principle. More "semi-reserved duplicate strands" are obtained by repeating the three processes of denaturation, annealing and extension, and the new strand becomes the template for the next cycle.

Single Nucleotide Polymorphism (SNP) refers mainly to DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. It is the most common one of the human heritable variants, accounting for over 90% of all known polymorphisms. SNP detection has a number of applications in medical diagnostics, such as for detecting genetic variations associated with genetic diseases, such as variations of the breast cancer genes BRCA1 and BRCA 2; can be used to predict the effect of certain drugs on the coagulation function of an individual, such as warfarin; may be used to assess the risk of an individual for certain diseases, such as cardiovascular disease, diabetes, and the like.

A liquid chip (also called as flow fluorescent technology) is a clinical application type high-flux luminous detection technology based on microsphere and flow technology, belongs to a novel biochip technology platform and consists of microsphere, probe molecule, molecule to be detected and reporter molecule. The core technology is that probes corresponding to different targets are coated on microspheres with different fluorescent codes, after a sample to be detected is added, the probe is combined with a reporter molecule with another fluorescent light to form a compound of the microsphere probe target reporter molecule, and a signal value is read in a flow fluorometer, so that multiple targets can be detected on one sample at the same time.

The existing detection methods for gene polymorphism generally comprise a sequencing method, a PCR probe method, a PCR-ARMS (polymerase chain reaction-amplified tandem mass spectrometry), a gene chip method, a nucleic acid mass spectrometry and the like, wherein the sequencing method, the PCR probe method and the PCR-ARMS have the problem of low detection flux and cannot meet the detection requirements of a plurality of gene polymorphisms; the detection flux of the gene chip and the nucleic acid mass spectrometry method is higher than that of the former few, but the operation time is too long, the steps are complicated, and specific consumables are needed to be relied on.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, an object of the present application is to provide a liquid chip-based SNP detection method for solving the above-described problems.

To achieve the above and other related objects, according to one aspect of the present application, there is provided a liquid chip-based SNP detection primer comprising an upstream primer and a downstream primer; the upstream primer sequentially comprises a tag sequence, a partition wall modification, a first specific sequence and a first enzyme cutting sequence from a 5 'end to a 3' end; the downstream primer sequentially comprises a detection group, a second specific sequence and a second enzyme cutting sequence from the 5 'end to the 3' end, wherein the first enzyme cutting sequence or the second enzyme cutting sequence contains RNA bases matched with SNP loci of target genes.

The second aspect of the application provides a liquid chip-based SNP detection method, comprising the following steps:

1) Carrying out polymerase chain reaction with a target gene by adopting the SNP detection primer, wherein RNA shearing enzyme is added into a polymerase chain reaction system to obtain a double-stranded DNA product with biotin and tag sequences;

2) Carrying out hybridization reaction on the microsphere with the anti-tag sequence and the double-stranded DNA product in the step 1) to obtain a hybridization product;

3) And adding a color reagent into the hybridization product, incubating, detecting a signal, and analyzing to obtain a detection result.

The third aspect of the application provides a gene detection kit, which comprises the SNP detection primer, and optionally one or more of microspheres with anti-tag sequences and RNA shearing enzymes.

In a fourth aspect, the present application provides the use of the aforementioned SNP detection primer, the aforementioned SNP detection method, or the aforementioned gene detection kit in SNP detection.

Compared with the prior art, the application has the beneficial effects that:

1. According to the application, the enzyme cutting sequence is designed on the primer, and the RNA base in the primer can be cut by RNase enzyme only after the hybridization of the upstream primer and the downstream primer with the completely matched target sequence, so that normal extension can be realized, thereby reducing primer dimer and other non-specific amplification products, and realizing single-tube multiplex amplification of PCR; the flow fluorescence can detect a plurality of targets through the quantum dot microsphere, and can realize the simultaneous detection of a plurality of SNP loci.

2. The application provides a SNP detection method based on a liquid chip technology, which combines the multiple PCR technology, constant temperature hybridization based on the liquid chip technology, detection technology, specific sequence and coding microsphere principle, designs a gene detection kit for warfarin drug guidance, can realize that CYP2C9 and VKORC1 gene loci obtain double-stranded DNA products after the enrichment of multiple PCR target sequences, and realizes hybridization and signal detection of a probe system under the constant temperature condition.

3. The application solves the problem that multi-target detection is needed in gene polymorphism detection, can realize the amplification and detection of the same tube, uses consumable materials which are all universal consumable materials in the market, and saves time and cost.

Drawings

FIG. 1 is a flow chart showing the detection principle and steps of the present application.

FIG. 2 is a two-dimensional graph of genotyping in an embodiment of the application. Wherein, number 1 represents CYP2C9 x 3AA, number 2 represents CYP2C9 x 3CC, number 3 represents CYP2C9 x 3AC, number 4 represents VKORC1 GG, number 5 represents VKORC1AA, number 6 represents VKORC1 GA, number 7 represents NTC, and number 8 represents NTC. By setting four corner lines, 4 regions can be intuitively obtained, wherein the upper left corner represents pure and mutant, the lower left corner represents negative control, the upper right corner represents heterozygous mutant, and the lower right corner represents wild type.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application will be further described with reference to examples. It is to be understood that the examples are provided for the purpose of illustrating the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventor of the present application has found a liquid chip-based SNP detection method through a great deal of research and study, and completed the present application on this basis.

The application provides a SNP detection primer based on a liquid chip, which comprises an upstream primer and a downstream primer; the upstream primer sequentially comprises a tag sequence, a partition wall modification, a first specific sequence and a first enzyme cutting sequence from a 5 'end to a 3' end; the downstream primer sequentially comprises a detection group, a second specific sequence and a second enzyme cutting sequence from the 5 'end to the 3' end, wherein the first enzyme cutting sequence or the second enzyme cutting sequence contains RNA bases matched with SNP loci of target genes. Matching in this context means identical or complementary to the target gene or target site.

In the SNP detection primer provided by the application, a first digestion sequence sequentially comprises a first RNA base, a third specific sequence, a mismatched base and 3' block modification from a 5' end to a 3' end. The second cleavage sequence comprises a second RNA base, a fourth specific sequence, mismatched bases and 3' block modification in sequence from the 5' end to the 3' end. The first specific sequence and/or the third specific sequence is adapted to specifically bind to a first strand of a target gene; the second specific sequence and/or the fourth specific sequence is adapted to specifically bind to the second strand of the target gene. The first strand and the second strand of the target gene are complementarily paired. Either the first RNA base or the second RNA base matches the SNP site. Because RNA base in the primer can be cut by RNaseH enzyme only after the upstream primer and the downstream primer are hybridized with the completely matched target sequence, and can be extended normally, primer dimers and other non-specific amplification products are reduced, and single-tube multiplex amplification of PCR is realized; the flow fluorescence can detect a plurality of targets through the quantum dot microsphere, and can realize the simultaneous detection of a plurality of SNP loci. The third specific base is used to match with a partial sequence of the target gene.

In the SNP detection primer provided by the application, mismatched bases are bases different from bases at positions corresponding to a target gene. For example, when the base at the corresponding position is C, the mismatched base may be any one of A, G, T. The 3' block modification includes one or a combination of C3Spacer, C6 Spacer, ddSpacer. C3Spacer is a phosphoramidite, and Spacer C3 is often placed at the 3' end of an oligonucleotide as a blocking group for oligonucleotide extension, while Spacer C3 is resistant to exonucleases. The 3' block modification serves as a blocking group preventing extension of the oligonucleotide. The synergistic effect of the third specific base, the mismatched base and the 3' block modification can reduce the nonspecific amplification of the primer, thereby improving the specificity of the primer and ensuring more accurate amplification effect.

In the SNP detection primer provided by the application, the target gene is selected from CYP2C9 or VKORC1. Among them, CYP2C9 belongs to the cytochrome P450 superfamily, and CYP2C9 can hydroxylate drugs with many different properties, mainly acidic substrates. The CYP2C9 gene is located on human chromosome 10q24.2, has a total length of about 50.71kb, and has 9 exons and 8 introns. It has been found to date that CYP2C9 has multiple mutant alleles of CYP2C 9-CYP 2C9, most commonly wild-type CYP2C9 x 1 and mutant CYP2C9 x2 and CYP2C9 x 3, other mutants except for CYP2C9 x 13, all found in a single ethnic group, with less related studies, most studied currently being CYP2C9 x2 and CYP2C9 x 3.CYP2C9 x2 mutation is a mutation of C430> T on exon 3, which causes Arg144> Cys144 amino acid substitution, CYP2C9 x 3 is a mutation of A1075> C on exon 7, which causes Ile359> Leu359 amino acid substitution. The vitamin epoxide reductase complex 1 (VKORC 1) gene is the rate-limiting enzyme for vitamin K dependent clotting factor production. Warfarin exerts a pharmaceutical effect by inhibiting this enzyme to influence vitamin K to participate in the catalytic process of carboxylase. There are a large number of polymorphic sites in the VKORC1 coding and non-coding regions, where variants at positions 1639G > A and 1173C > T affect warfarin application doses. CYP2C9 and VKORC1 can be used for explaining the individual drug variability of warfarin due to their own gene polymorphism, and are decisive factors for the individual drug variability.

In the SNP detection primer provided by the application, the tag sequence is a 10-30 bp sequence of ATCG random combination. the tag sequence can be complementarily paired with the anti-tag sequence on the microsphere, and is used for capturing double-stranded DNA products amplified by polymerase chain reaction by the microsphere.

In the SNP detection primer provided by the application, the partition wall modification comprises dSpacer. dSpacer is tetrahydrofuran, and addition of dSpacer to an oligonucleotide can introduce a stable abasic site. The partition modification provides the necessary spacing to reduce the interaction between the labeling group and the oligonucleotide. The modification of the partition wall is introduced into the oligonucleotide, usually in order to establish a distance between the oligonucleotide or between the oligonucleotide and other functional groups, to avoid steric hindrance, to reduce adverse interactions between the groups, to increase flexibility, etc.

In the SNP detection primer provided by the application, the detection group comprises biotin. The very stable, non-covalent interaction between avidin and biotin is one of the most commonly used tools in the chemical and biological fields. Biotin is a constituent of vitamin B2 complex. It has high affinity and binding force with chicken albumin, avidin, mycoprotein and streptavidin. Both avidin and streptavidin are tetrameric proteins that bind tightly to four molecules of D-biotin.

In the SNP detection primer provided by the application, the upstream primer comprises 1 wild upstream primer and 2-3 mutant upstream primers. The upstream primer contains RNA bases that match the SNP site. SNP refers to single base mutation, comprising a wild base and a mutant base, wherein the RNA base of the wild type upstream primer is matched with the wild base of the SNP locus, and the RNA base of the mutant upstream primer is matched with the mutant base of the SNP locus. In one embodiment of the present application, when the SNP site is A > C, the RNA base of the wild-type upstream primer is A and the RNA base of the mutant upstream primer is C. In some embodiments, the tag sequence of the wild-type upstream primer is different from the tag of the mutant upstream primer in order to detect wild-type, mutant, and hybrid, respectively, by different microspheres. The wild type and mutant forms of the present application refer to homozygotes, such as AA or CC, and heterozygotes are AC unless otherwise specified.

In the SNP detection primer provided by the application, when a target gene is CYP2C9 x 3, the sequence of the wild upstream primer is shown as SEQ ID NO:1, the sequence of the mutant upstream primer is shown as SEQ ID NO:2, the downstream primer is shown as SEQ ID NO: 3. When the target gene is VKORC1, the sequence of the wild type upstream primer is shown in SEQ ID NO:4, the sequence of the mutant upstream primer is shown as SEQ ID NO:5, the downstream primer is shown as SEQ ID NO: shown at 6.

In another aspect, the application provides a liquid chip-based SNP detection method, comprising the following steps:

1) Carrying out polymerase chain reaction with a target gene by adopting the SNP detection primer, and adding RNA shearing enzyme into a polymerase chain reaction system to obtain a double-stranded DNA product with biotin and tag sequences;

The SNP detection method of the application is for the purpose of non-disease diagnosis or treatment.

In the SNP detection method provided by the application, step 1) refers to the step of carrying out polymerase chain reaction with a target gene by adopting the SNP detection primer, wherein RNA shearing enzyme is added into a polymerase chain reaction system, so as to obtain a double-chain DNA product with biotin and tag sequences. Wherein the RNA cleaving enzyme comprises RNase HII enzyme. In the PCR reaction, after the upstream primer and the downstream primer are in specific complementary pairing with the sequence of a target gene, RNA bases on the upstream and the downstream can be recognized and cut by RNase HII enzyme, so that a 3 '-end enzyme cutting sequence is cut off, and a specific sequence primer left after the cutting off keeps a 3' -end hydroxyl group, thereby enabling the primer to normally extend for the PCR reaction, and the obtained amplicon simultaneously contains a tag sequence and double-stranded nucleic acid with a biotin modification, can be captured by a microsphere connected with an anti-tag, and is detected through the reaction of a corresponding color developing agent and the biotin.

In the SNP detection method provided by the application, the step 2) is to carry out hybridization reaction between the microsphere with the anti-tag sequence and the double-stranded DNA product of the step 1) to obtain a hybridization product. Wherein the microsphere is selected from fluorescent microsphere, quantum dot microsphere or other conventional microsphere with encodable function. When the target gene is CYP2C9 x 3, the wild homozygote (AA) anti-tag sequence is shown in SEQ ID NO:7, the mutant homozygote (CC) anti-tag sequence is shown as SEQ ID NO: shown at 8. When the target gene is VKORC1, the wild-type homozygote (GG) anti-tag sequence is shown in SEQ ID NO:9, the mutant homozygote (AA) anti-tag sequence is shown as SEQ ID NO: shown at 10.

In the SNP detection method provided by the application, step 3) is to add a color developing agent into the hybridization product, incubate, detect signals and analyze to obtain detection results. Wherein the color-developing agent reacts with biotin. In one embodiment of the application, the chromogenic agent is SA-PE (streptavidin, R-phycoerythrin conjugate), wherein streptavidin is a biotin-binding protein of bacterial origin, rhodophyta Phycoerythrin (RPE) has a molecular weight of 240,000 daltons and a quantum efficiency of 78%, and is an extremely bright reagent that absorbs widely and emits bright orange signals making it an ideal choice for flow, microplate and microarray applications when sensitive detection of biotin is desired.

The application also provides a gene detection kit, which comprises the SNP detection primer, and optionally one or more of microspheres with anti-tag sequences and RNA shearing enzymes. In the PCR reaction, after the specific sequence is specifically complementary and paired with the target sequence, RNA bases at the upstream and downstream of the detection primer can be recognized and cut by RNA shearing enzyme such as RNase HII enzyme, so that the 3 '-end enzyme cutting sequence is cut off, the specific sequence primer left after the cutting off keeps 3' -end hydroxyl, the primer can be normally extended for the PCR reaction, and the obtained amplicon simultaneously contains the labeled tag sequence and double-stranded nucleic acid with biotin modification, can be captured by the microsphere connected with anti-tag, and can be detected through the reaction of the corresponding color developing agent and the biotin.

In another aspect, the application provides the aforementioned SNP detection primer, the aforementioned SNP detection method, or the aforementioned gene detection kit for use in SNP detection. The SNP detection of the present application is for non-disease diagnosis or treatment purposes. The application solves the problem that multi-target detection is needed in gene polymorphism detection, can realize the amplification and detection of the same tube, uses consumable materials which are all universal consumable materials in the market, and saves time and cost.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application.

Example 1

The embodiment provides a SNP detection method based on a liquid chip technology, which combines the multiple PCR technology, constant temperature hybridization based on the liquid chip technology, a detection technology, a specific sequence and a coding microsphere principle, designs a gene detection kit for warfarin drug guidance, can realize that CYP2C9 and VKORC1 gene loci obtain double-stranded DNA products after the enrichment of multiple PCR target sequences, and realizes hybridization and signal detection of a probe system under the constant temperature condition.

1. CYP2C9 and VKORC1 templates (templates are DNA sequences synthesized according to NCBI search) were amplified by using 1 primer with a label and 2 specific primers (specific primer sequences are shown in Table 1) connected with different tag sequences, PCR procedures are shown in Table 2, and PCR systems are formulated as shown in Table 3 to obtain detection target double-stranded DNA products of CYP2C9 and VKORC1 with biotin labels and free tag sequences (FIG. 1-PCR part).

Table 1 PCR primers

Table 2 PCR System formulation

Wherein RNase HII is available from IDT-11-02-12-01.F+R represents the upstream primer and the downstream primer at a concentration of 10. Mu.M, and the total volume of both primers is 2.5. Mu.L.

Table 3PCR procedure

2. The single-stranded DNA probes for differentiating between the different types of CYP2C9 and VKORC1 are attached to the microspheres by chemical reaction to form covalent bonds, a procedure consistent with the microsphere treatment in published patent 202110992820.3. The probe shown in Table 4 comprises a nucleic acid sequence complementary to the free tag sequence (anti-tag) attached to the double-stranded product sequence with the tag in step 1

TABLE 4 probe sequences

3. The microspheres with anti-tag in step 2 were added to the double-stranded amplification product with biotin tag and tag sequence in step 1, and subjected to constant temperature hybridization, the hybridization procedure being as shown in Table 5.

TABLE 5 hybridization procedure

Step (a)	Temperature (. Degree. C.)	Time (min)	Cycle number
				Hybridization	50	20	1

4. To the hybridization product in step 3, a chromogenic agent (SA-PE, available from Invitrogen C27206 in this example) was added, incubated at constant temperature, and the incubation procedure was as shown in Table 6.

Table 6 incubation procedure

Step (a)	Temperature (. Degree. C.)	Time (min)	Cycle number
				Incubation	50	10	1

5. And (3) analyzing the result of the incubation product in the step (4) by using a flow cytometry analyzer. The test results are shown in Table 7. Wherein NTC refers to negative control in which the template is water.

TABLE 7 hybridization Signal

Numbering device	Sample name	Wild probe	Mutant probes
				1	CYP2C9*3AA	196101	78136.2
2	CYP2C9*3CC	77409.2	163818.4
				3	CYP2C9*3AC	127748	125231.1
4	VKORC1 GG	190822.1	71320.6
				5	VKORC1 AA	83797.3	171794.3
6	VKORC1 GA	125712.5	131144.6
				7	NTC	7005.2	5064.2
8	NTC	11388.5	5058.1

As can be seen from the results of Table 7 or FIG. 2, the signal value of hybridization of the amplification product corresponding to each template clearly distinguishes the wild type, pure and mutant and heterozygous mutant results of the corresponding sample added. The PCR product obtained by the multiplex PCR amplification mode can realize that the tag sequence on the product is effectively hybridized with the probe to obtain a hybridization signal, and can effectively judge the typing of the tested sample. Through the design of block primers, SNP loci which are specifically combined are successfully hydrolyzed under the action of RNaseHII enzyme, a reaction hole with a template and a reaction hole without a template (NTC) are well distinguished, and the multiplex PCR process is successfully completed.

Comparative example 1

The CYP2C9 x 3 (1075A > C) locus is tested by designing an experimental comparison common primer, and the primer arrangement is consistent with other conditions except for no enzyme cutting locus, and the detailed comparison primer table in Table 8 is shown.

Table 8 comparative primer Table

The PCR system was prepared by referring to Table 9 using the above primers, and experiments were performed in parallel by setting the primers in Table 1 and referring to Table 2.

Table 9 PCR System formulation

System composition	25μL
		2×Taq Mix	12.5
Template	5
		F+R	2.5
ddH₂O	5

2. The subsequent hybridization and detection procedures were the same as in step 2-4 of example 1.

3. Results

TABLE 10 hybridization Signal

As can be seen from the above table, if the primers of common design (primers in Table 8) are used, the background (No. 8) signal will be higher, probably because the upstream and downstream primers form primer dimers, and the probes with labeling effect and biotin for detection will be captured by the microspheres and the signal will be detected; secondly, for sample detection, the wild type and the heterozygous type (numbers 5 and 7) cannot be distinguished from the signal level, probably because the mutant primer also generates nonspecific amplification in the wild type sample. Therefore, the primer design in Table 1 gave far better results than the primer design in Table 8.

The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A SNP detection primer based on a liquid chip, which is characterized by comprising an upstream primer and a downstream primer; the upstream primer sequentially comprises a tag sequence, a partition wall modification, a first specific sequence and a first enzyme cutting sequence from a5 'end to a 3' end; the downstream primer sequentially comprises a detection group, a second specific sequence and a second enzyme cutting sequence from the 5 'end to the 3' end, wherein the first enzyme cutting sequence or the second enzyme cutting sequence contains RNA bases matched with SNP loci of target genes.

2. The liquid chip-based SNP detection primer of claim 1, wherein the first cleavage sequence comprises, in order from the 5' end to the 3' end, a first RNA base, a third specific sequence, a mismatched base, and a 3' block modification; the second digestion sequence comprises a second RNA base, a fourth specific sequence, mismatched bases and 3' block modification from the 5' end to the 3' end in sequence; the first specific sequence and/or the third specific sequence is adapted to specifically bind to a first strand of a gene of interest; the second specific sequence and/or the fourth specific sequence is/are suitable for specifically binding to a second strand of a target gene, and the first RNA base or the second RNA base is matched with an SNP locus;

and/or the target gene is selected from CYP2C9 or VKORC1.

3. The SNP detection primer based on liquid chip as set forth in claim 1, wherein,

The tag sequence is a random DNA sequence of 10-30 bp;

and/or, the partition modification comprises dSpacer;

and/or, the detection group comprises biotin;

And/or, the 3' block modification comprises a combination of one or more of C3 Spacer, C6 Spacer, ddSpacer.

4. The liquid chip-based SNP detection primer of claim 1 wherein the upstream primer comprises an RNA base matching the SNP site and comprises 1 wild-type upstream primer and 2-3 mutant upstream primers.

5. The liquid chip-based SNP detection primer of claim 4, wherein the RNA base of the wild-type upstream primer matches the wild base of the SNP site and the RNA base of the mutant upstream primer matches the mutant base of the SNP site.

6. The liquid chip-based SNP detection primer of any one of claims 1-5, wherein the detection primer has a sequence as set forth in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. or SEQ ID NO: shown at 6.

7. The SNP detection method based on the liquid chip is characterized by comprising the following steps:

1) Carrying out polymerase chain reaction on the SNP detection primer according to any one of claims 1 to 6 and a target gene, wherein RNA shearing enzyme is added into a polymerase chain reaction system to obtain a double-stranded DNA product with biotin and tag sequences;

8. The SNP detection method of claim 7 comprising a combination of one or more of the following features: a1 In step 1), the RNA cleaving enzyme is RNase HII enzyme;

a2 In step 2), the microspheres are selected from fluorescent microspheres or quantum dot microspheres;

a3 In the step 2), the anti-tag sequence is shown as SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. or SEQ ID NO:10 is shown in the figure;

a4 In step 3), the color-developer reacts with biotin.

9. A gene detection kit comprising the SNP detection primer according to any one of claims 1 to 6, and optionally, one or more of an anti-tag sequence-carrying microsphere and an RNA shearing enzyme.

10. The use of the SNP detection primer according to any one of claims 1 to 6, the SNP detection method according to any one of claims 7 to 8, or the gene detection kit according to claim 9 in SNP detection.