CN116426650A - Primer, kit, detection method and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular biology, and provides a primer, a kit and a detection method for detecting the rs396991 locus polymorphism of a CD16A gene and application thereof. The primer comprises: first primer set for detecting rs396991 site polymorphism: 5'-acagcggctcctacttctgcagggggcCtt-3' and 5' -gtctctgaagacacatttttactcTcGac-3, and a second primer pair for amplifying the CD16A gene sequence: 5'-gagaataagctctggcgatac-3' and 5'-gcacctcggttacttcatctgta-3'. The invention establishes a method for rapidly detecting the rs396991 locus polymorphism of the CD16A gene by a one-step method, and has the advantages of simple operation, high accuracy, good specificity and the like.

Description

Primer, kit, detection method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a primer for detecting rs396991 locus polymorphism of a CD16A gene, a kit and a detection method and application thereof.

Background

NK cells show great potential in tumor therapy, applications including checkpoint blockade, ADCC-enhanced antibodies, agonist antibodies and multi-specific NK cell agonists, as well as adoptive NK cell therapies, etc., which significantly expand the scope of clinically selected therapeutic approaches for patients. Human IgG fcγiiia (also known as FCGR3A or CD 16A) is the only fcγr receptor expressed by NK cells, which binds to IgG attached to target cells, promoting antibody-dependent cell-mediated cytotoxicity (ADCC). The polymorphism of CD16A158 Val/Phe (site rs 396991) affects the affinity of the FcgammaR receptor for binding IgG, and CD16A158V has higher affinity for IgG1 than the receptor encoded by CD16A158F, and patients expressing the high affinity CD16A158V (valine) allele have better clinical efficacy than patients expressing the low affinity CD16A158F (phenylalanine) allele and the F/F genotype. Currently, low affinity variants in the population are dominant alleles, with only 31% of the CD16a158V population and as low as 19.2% in asian populations (data from NCBI-SNP database rs 396991).

Due to the high homology between fcγriiia (also known as FCGR3A or CD 16A) and fcγriiib (also known as FCGR3B or CD 16B), CD16A has two dominant alleles (158V or 158F); CD16B has two major alleles, human neutrophil antigen 1 (NA 1) and NA2, with four polymorphic amino acids. Only two amino acid differences were used to distinguish between CD16A and CD16B. Wherein the sequence identity of CD16A-158V and CD16B-NA2 is greater than 97% and only four amino acid residues differ in the extracellular antibody binding domain.

Therefore, the polymorphism identification of the rs396991 locus of the CD16A gene is carried out by utilizing a detection means with simple operation, high accuracy and good specificity, and is important for promoting the research and development of NK cell products and clinical application.

Disclosure of Invention

The invention aims to overcome the problems in the prior art and provides a primer for detecting the rs396991 locus polymorphism of a CD16A gene, a kit containing the primer and a method for detecting the rs396991 locus polymorphism of the CD16A gene. The primer for detecting the rs396991 locus polymorphism of the CD16A gene can rapidly judge genotype CD16A158F/F, CD A158F/V and CD16A158V/V from a specific band in an electrophoresis chart of a PCR product only by one-step PCR reaction (namely a one-step method), and has the advantages of simple operation, high accuracy, good specificity and the like.

In order to achieve the above object, the first aspect of the present invention provides a primer comprising a first primer set for detecting a polymorphism at the rs396991 locus of the CD16A gene;

the first primer set includes:

CD16A-158F：5’-acagcggctcctacttctgcagggggcttt-3’(SEQ ID NO:1)，

CD16A-158V：5’-tctctgaagacacatttttactcccaac-3’(SEQ ID NO:2)，

in the CD16A-158F and the CD16A-158V, at least 1 mismatched base is introduced within 5 bases near the 3' end except for the first base.

The 158 th amino acid coded by the CD16A gene can be Val or Phe, namely, polymorphism of the rs396991 site. Wherein, the codon corresponding to Val (valine, abbreviated as V) is GTT, and the codon corresponding to Phe (phenylalanine, abbreviated as F) is TTT.

In the invention, the 'CD 16A-158F' is a primer for detecting the TTT at the site rs396991, and can also be called as an 'Inner primer-F'; the "CD16A-158V" is a primer for detecting the GTT at the site of rs396991, and can also be called as an "Inner primer-R (Inner-R)".

In the two primers CD16A-158F and CD16A-158V for detecting the polymorphism of the locus rs396991 of the CD16A gene, the 3' tail end of the primer is positioned on the locus rs396991 of the CD16A gene, and the two primers are opposite in direction and belong to different genotypes.

The inventor of the invention discovers that introducing at least 1 mismatched base except the first base in 5 bases close to the 3' end of the primer for detecting the polymorphism of the rs396991 locus of the CD16A gene can reduce the amplification efficiency of a non-target sequence, improve the specificity of PCR amplification and further improve the accuracy of detecting the polymorphism of the rs396991 locus of the CD16A gene.

In one example, the sites where mismatched bases are introduced are not adjacent.

In the invention, in the CD16A-158F and the CD16A-158V primer, the risk of off-target is smaller when the introduced mismatched base loci are not adjacent, and the detection accuracy is higher.

In one example, the first primer set includes:

CD16A-158F：5’-acagcggctcctacttctgcagggggcv ₁ tt-3’(SEQ ID NO:3)，

CD16A-158V：5’-tctctgaagacacatttttactcd ₁ cb ₁ ac-3 '(SEQ ID NO: 4) and/or 5' -tctctctgaagacattttactctcccb ₂ ac-3’(SEQ ID NO:5)，

Wherein the v ₁ At least one of a, c or g; said b ₁ At least one of t, c or g; said d ₁ At least one of a, t or g; said b ₂ At least one of t, c or g. v ₁ ，b ₁ ，d ₁ And b ₂ Is the mismatched base introduced.

The v is as follows ₁ Is at least one of a, c or g, and is v ₁ Is a, v ₁ For c and v ₁ There may be only one kind of primers differing in g of three nucleotide sequences, there may be a mixture of two kinds, or there may be a mixture of three kinds.

The b is that ₂ At least one of t, c or g, b ₂ T, b ₂ Is c and b ₂ There may be only one kind of primers differing in g of three nucleotide sequences, there may be a mixture of two kinds, or there may be a mixture of three kinds.

When two mismatched bases are introduced, the b ₁ At least one of t, c or g, d ₁ At least one of a, t or g may be a primer sequence alone or a plurality of primer combinations having different nucleic acid sequences, and the primer combinations having different nucleotide sequences may be selected according to random arrangement combinations.

In one example, in the first primer set, CD16A-158F and CD16A-158V may be primer combinations that each independently include 1, 2, 3, or more than 3 nucleic acid sequences. For example, when a mismatched base is introduced, there may be several combinations of:

(1) V in CD16A-158F primer ₁ Is c base, b in the CD16A-158V primer ₂ Is c base;

(2) CD16A-158F primer v ₁ Is a combination of two primers of c base and a base, and the CD16A-158V primer is b ₂ Is a combination of two primers of c base and t base;

(3) CD16A-158F primer v ₁ Three primer combinations of c base, a base and g base, and the CD16A-158V primer is b ₂ Is a combination of three primers, c, t and g bases.

The other cases of introducing two mismatched bases and introducing three mismatched bases are similar to the above case of introducing one mismatched base, and both CD16A.about.158F and CD16A.about.158V may be primer combinations having different nucleic acid sequences, and how the primer sequences are combined is not particularly limited as long as the primer or the target band amplified by the primer combination is the same.

In one example, the first primer set for detecting a polymorphism at the rs396991 locus of the CD16A gene comprises:

CD16A-158F-F1：5’-acagcggctcctacttctgcagggggcCtt-3’(SEQ ID NO:6)，CD16A-158V-R1：5’-gtctctgaagacacatttttactcTcGac-3' (SEQ ID NO: 7); and/or CD16A-158V-R1':5' -tctctgaagacacatttttactcccCac-3' (SEQ ID NO: 8); in the above primers, the base represented by the capital letter (underlined) is the base to which the mismatch was introduced.

In a preferred embodiment, the first primer set for detecting a polymorphism at the rs396991 locus of the CD16A gene comprises: CD16A-158F-F1:5' -acagcggctcctacttctgcagggggcCtt-3' (SEQ ID NO: 6), and CD16A-158V-R1:5' -gtctctgaagacacatttttactcTcGac-3' (SEQ ID NO: 7), wherein the base represented by the capital letter (underlined) in the above primer is a base to which a mismatch is introduced.

In one example, the first primer set for detecting a polymorphism at the rs396991 locus of the CD16A gene comprises: CD16A-158F-F1:5' -acagcggctcctacttctgcagggggcCtt-3 '(SEQ ID NO: 6), and CD16A-158V-R1':5' -tctctgaagacacatttttactcccCac-3’(SEQ ID NO:8) In the above primer, the base represented by capital letters (underlined) is a base to which a mismatch is introduced.

The invention also provides a second primer pair for amplifying a CD16A gene sequence containing rs396991 locus polymorphism; the second primer pair comprises a forward primer and a reverse primer, wherein the distance between the forward primer and the rs396991 locus polymorphism is not equal to the distance between the reverse primer and the rs396991 locus polymorphism.

In one example, the forward primer is at a distance from the rs396991 site polymorphism that differs from the reverse primer by no less than 50bp, preferably no less than 100bp from the rs396991 site polymorphism.

By limiting the difference of the distance between the forward primer and the reverse primer and the polymorphism of the rs396991 locus of the CD16A gene, the sizes of PCR products amplified by adopting the first primer group and the second primer group are different, and the PCR products can be distinguished in an electrophoresis strip of the PCR products more easily, so that the genotype of the rs396991 locus of the CD16A gene can be conveniently judged.

In one example, the second primer pair comprises:

CD16A-F1：5’-gagaataagctctggcgatac-3’(SEQ ID NO:9)，

CD16A-R1:5'-gcacctcggttacttcatctgta-3' (SEQ ID NO: 10); and/or the number of the groups of groups,

CD16A-F2：5’-gagaataagctctggcgatac-3’(SEQ ID NO:9)，

CD16A-R2:5'-ctttgatgtgaccttagggaaact-3' (SEQ ID NO: 11); and/or the number of the groups of groups,

CD16A-F3：5’-gccaccgtcaccttattcct-3’(SEQ ID NO:12)，

CD16A-R3:5'-ttgcaggttccacacacagg-3' (SEQ ID NO: 13); and/or the number of the groups of groups,

CD16A-F4：5’-ggtcttggtatgacttcagttc-3’(SEQ ID NO:14)，

CD16A-R4：5’-ttgcaggttccacacacagg-3’(SEQ ID NO:13)；

wherein, the "CD16A-F" represents the nucleotide sequence of the forward primer; "CD16A-R" represents the nucleotide sequence of the reverse primer. The 4 primer pairs can specifically amplify the CD16A gene sequence containing the rs396991 locus. The "CD16A-F" and "CD16A-R" may also be referred to as "outer primer-F" and "outer primer-R", respectively.

In one example, the second primer pair is CD16A-F1:5'-gagaataagctctggcgatac-3' (SEQ ID NO: 9) and CD16A-R1:5'-gcacctcggttacttcatctgta-3' (SEQ ID NO: 10). The primer pair can specifically amplify a CD16A gene sequence containing an rs396991 locus, and the full length of a PCR product fragment obtained by amplification is 1583bp.

In one example, the primers for detecting the rs396991 locus polymorphism of the CD16A gene comprise:

second primer pair for amplifying CD16A gene sequence: CD16A-F1:5'-gagaataagctctggcgatac-3' (SEQ ID NO: 9) and CD16A-R1:5'-gcacctcggttacttcatctgta-3' (SEQ ID NO: 10);

and a first primer set for detecting an rs396991 locus polymorphism: CD16A-158F-F1:5' -acagcggctcctacttctgcagggggcCtt-3' (SEQ ID NO: 6), and CD16A-158V-R1:5' -gtctctgaagacacatttttactcTcGac-3’(SEQ ID NO:7)。

Referring to the reaction principle schematic diagram of fig. 1, in the PCR reaction process, if the four primers are completely matched with the template, three products with different lengths can be amplified (if the two inner primers are located on the same site, no long fragment amplified product is formed); if one of the two inner primers is not matched with the template, two PCR products of different lengths can be formed. Therefore, the genotype of the locus rs396991 can be judged according to the size of the PCR product after electrophoresis.

The second aspect of the invention provides the application of the primer in the first aspect of the invention in detecting the rs396991 locus polymorphism of the CD16A gene.

In such applications, the primers provided in the first aspect of the invention may be used to determine the genotype of the rs396991 locus of the CD16A gene in combination with molecular biological methods conventionally used in the art (e.g., PCR, gene sequencing, etc.).

The third aspect of the invention provides a kit for detecting CD16A gene polymorphism, which comprises the primer for detecting CD16A gene polymorphism according to the first aspect of the invention.

In one example, the kit further comprises a DNA polymerase that does not have 3' terminal calibration modification functionality. When the primer for detecting the polymorphism of the CD16A gene is used for PCR amplification, DNA polymerase which does not have 3' end correction and modification functions is used in a matched mode, mismatched bases introduced into the primer can be prevented from being repaired, the function of mismatched bases is reserved, and the specificity of the primer is improved.

In one example, the DNA polymerase without 3' end correction modification may be Taq enzyme without base mismatch modification.

In one example, the kit further comprises reagents used for PCR amplification, such as dNTP mixtures, mgCl ₂ And buffer systems, etc.

In a fourth aspect, the invention provides a method for detecting the rs396991 locus polymorphism of a CD16A gene, comprising the following steps: and (3) performing PCR reaction by using the primer disclosed in the first aspect of the invention or the kit disclosed in the third aspect of the invention, and judging the rs396991 locus polymorphism of the CD16A gene according to the PCR product.

In the invention, after the PCR reaction is carried out by utilizing the primer or the kit, the obtained PCR product can be directly subjected to gel electrophoresis, and the genotype of the site rs396991 can be directly judged according to the size of an electrophoresis band; the PCR product can also be directly subjected to gene sequencing to judge the genotype of the locus rs 396991.

In one example, the method for detecting the rs396991 locus polymorphism of the CD16A gene comprises the following steps:

s1: preparing a sample to be tested;

s2: taking the sample to be detected as a template, and carrying out PCR reaction by adopting the primer or the kit to obtain a PCR product;

s3: and (3) carrying out electrophoresis on the PCR product, and judging the polymorphism of the CD16A gene according to the electrophoresis band.

The judgment of the polymorphism of the CD16A gene according to the electrophoresis band is specifically as follows:

if three bands appear in the PCR electrophoresis result and the size result is basically consistent with the 158F/V genotype, judging that the genotype of the rs396991 locus is F/V;

if the PCR electrophoresis result shows two bands and the size result is basically consistent with the band size result of the CD16A158F/F genotype, judging that the genotype of the locus rs396991 is F/F;

if the PCR electrophoresis result shows two bands and the size of the two bands is basically consistent with that of the CD16A158V/V genotype, judging that the genotype of the locus rs396991 is V/V.

In one example, the sample to be tested may be extracted genomic DNA, or other sample that may contain DNA.

In one example, the PCR reaction is an amplification block mutation system PCR (Tetra-primer amplification refractory mutation system PCR, tetra-primer ARMS-PCR).

The invention establishes a method for rapidly detecting the rs396991 locus polymorphism of the CD16A gene by a one-step method based on four primers Tetra-primer ARMS-PCR, and can rapidly judge the genotype of the rs396991 locus by a one-step PCR reaction (namely a one-step method) and a specific DNA band in an electrophoresis chart.

The method for detecting the CD16A gene polymorphism can simultaneously detect 96 samples by only taking 90 minutes through one common PCR reaction program, is very suitable for screening a large number of samples of CD16A genes, is not limited by high-cost instruments and equipment, and has the advantages of low cost, short time consumption and high efficiency.

In the method for detecting the polymorphism of the CD16A gene, the PCR reaction conditions can be adjusted according to actual needs and are not particularly limited.

In one example, the PCR system is shown in the following table:

component (A)
		PCR Master Mix(2×)	5μL
Template
		1 mu L (genomic DNA:20-50 ng)
Primer premix			1.5-2.5μL
	ddH 2 O	Make up to 10. Mu.L system

Wherein, the PCR Master Mix contains DNA polymerase without 3' end correction modification function.

In one example, the procedure for the PCR reaction is shown in the following table:

wherein "/" represents no cycle number.

In one example, the concentration of the primer is 0.3. Mu.M to 0.6. Mu.M. For example, 0.3. Mu.M, 0.35. Mu.M, 0.4. Mu.M, 0.45. Mu.M, 0.5. Mu.M, 0.55. Mu.M, and 0.6. Mu.M can be used.

In a preferred embodiment, the concentration of the primer is 0.4. Mu.M to 0.5. Mu.M.

The technical scheme adopted by the invention has the following beneficial effects:

(1) The primer for detecting the polymorphism of the rs396991 locus of the CD16A gene provided by the invention can rapidly judge the genotype of the rs396991 locus from a specific DNA strip in an electrophoresis chart by only one PCR reaction, and has the advantages of simplicity and convenience in operation, high accuracy, good specificity and the like;

(2) The method for detecting the rs396991 locus polymorphism of the CD16A gene provided by the invention can be used for simultaneously detecting 96 samples through one-time common PCR reaction program, is very suitable for screening a large number of samples of CD16A genes, and is not limited by high instrument and equipment, and has the advantages of low cost, short time consumption and high efficiency.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Drawings

FIG. 1 is a schematic diagram showing the principle of PCR reaction for detecting the rs396991 locus polymorphism of the CD16A gene;

FIG. 2 is a diagram showing agarose gel electrophoresis of example 1 of the present invention;

FIG. 3 shows agarose gel electrophoresis patterns of examples 2 to 4 of the present invention;

FIG. 4 shows agarose gel electrophoresis patterns of examples 5 and 6 of the present invention;

FIG. 5 is a schematic diagram showing a partial result of sequencing the PCR products of samples No. 01-05 in the verification example of the present invention;

FIG. 6 is a schematic diagram showing a partial result of sequencing of PCR products of samples No. 06-10 in the verification example of the present invention.

Detailed Description

The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.

The technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The invention is described in detail below in connection with specific embodiments, which are intended to be illustrative rather than limiting.

Experimental example sequencing detection of sample genotype

Designing a pair of primers, amplifying a CD16A gene sequence containing an rs396991 locus, wherein the total length of the amplified fragment is 800bp, and the nucleotide sequence of a forward primer is as follows: 5'-gccaccgtcaccttattcct-3' (SEQ ID NO: 12), the reverse primer nucleotide sequence is: 5'-ttgcaggttccacacacagg-3' (SEQ ID NO: 13).

PCR amplification was performed using DNA genome samples No. 01-11 as templates, and the PCR products were sequenced under the conditions of example 1 described below, and the sequencing results are shown in FIGS. 5 and 6. The genotypes of samples numbered 01-11 were finally judged as shown in Table 1 below:

TABLE 1

Wherein the allele of the 158F/V genotype representing the site rs396991 is T/G type, the allele of the 158F/F genotype representing the site rs396991 is T/T type, and the allele of the 158V/V genotype representing the site rs396991 is G/G type.

Example 1

A method for detecting a CD16A gene rs396991 locus polymorphism, comprising the steps of:

(1) Samples with known genotypes and numbers 01-11 after the test in the above experimental examples were used as templates:

(2) Primer premix solutions were prepared with the proportions of the components shown in Table 2 below:

TABLE 2

(3) Preparing PCR premix solution (product number: next holy 10103ES 03) containing Taq DNA Polymerase, dNTP mixture and MgCl ₂ And an optimized buffer system. During PCR reaction, only template DNA and primer premix are added for amplification, so that the operation steps of the experiment are greatly simplified, and the PCR reaction system is shown in the following table 3:

TABLE 3 Table 3

Component (A)
		Hieff Unicon Taqman PCR Master Mix(2×)	5μL
Template DNA
		1 mu L (genomic DNA:20-50 ng)
Primer premix			1.9μL
	ddH 2 O	Make up to 10. Mu.L system

(4) PCR amplification was performed, and the amplification procedure is shown in Table 4 below:

TABLE 4 Table 4

(5) Results

The theoretical band sizes of the PCR products are shown in Table 5 below:

TABLE 5

The PCR products of 11 samples were subjected to 2.5% agarose gel electrophoresis, and the result of the electrophoresis is shown in FIG. 2.

As can be seen from the electrophoresis results of FIG. 2, lanes 01, 02, 05 and 08 each present three bands, which are substantially consistent with the band size results for the CD16A158F/V genotype in Table 5. 03 Lanes 04, 09 and 11 each present two bands, which are substantially consistent with the band size results for the CD16A158F/F genotype in Table 5. 06 Lanes 07 and 10 each present two bands, which are substantially consistent with the band size results for the CD16A158V/V genotype in Table 5.

Example 2

The procedure of example 1 was followed, with the main differences that the primers used were different, as shown in Table 6 below:

TABLE 6

Example 3

The procedure of example 1 was carried out with the main difference that the primers used were different, as shown in Table 7 below:

TABLE 7

Example 4

The procedure is as in example 1, with the main differences that the primers used are different, as shown in Table 8 below:

TABLE 8

The three primer sets provided in examples 2 to 4 were used for PCR amplification reaction using 3 samples of known genotypes (No. 3: genotype 158F/F; no. 4: genotype 158F/V; no. 9: genotype 158V/V) as templates, and the PCR reaction system was the same as that of example 1.

The theoretical band sizes of the PCR products of examples 2-4 are shown in Table 9 below:

TABLE 9

The electrophoresis results of examples 2 to 4 are shown in FIG. 3 (numbers (1), (2) and (3) in the figures correspond to the primers of examples 2 to 4, respectively), and the electrophoresis chart shows that only the primer provided in example 2 has a suspected target amplified band, but it is apparent that the primer provided in example 2 has a problem of insufficient amplification specificity.

In examples 2-4, the rs396991 site polymorphism of the CD16A gene could be detected more accurately only if the forward primer and the reverse primer for amplifying the CD16A gene were different, and the difference in the result of the electrophoresis bands was large, indicating that the specific primer for amplifying the CD16A gene and the specific primer for detecting the rs396991 site polymorphism were matched.

Example 5

With reference to example 2, the main difference is that the primers used are different (primers CD16A-158V, which detect GTT at position rs396991, are optimized and introduce 2 mismatched bases), as shown in Table 10 below:

table 10

Example 6

With reference to example 5, the main difference is that the primers used are different (the reverse primer CD16A-R for amplifying the CD16A gene is optimized), as shown in Table 11 below:

TABLE 11

The PCR reaction was performed according to the two sets of primer pairs provided in examples 5 and 6 using 4 samples of known genotypes (No. 4: genotype 158F/V; no. 5: genotype 158F/F; no. 10: genotype 158V/V; no. MS05: genotype 158V/V) as templates, and the PCR reaction conditions were the same as in example 1.

The theoretical band sizes of the PCR products of examples 5 and 6 are shown in Table 12 below:

table 12

In order to improve the specificity of primer amplification, the downstream primer for detecting the rs396991 locus polymorphism in example 5 is optimized, and a mismatched base is introduced on the basis of the corresponding primer in example 2 to obtain a CD16A-158V-R1 primer (the same as in example 1); the downstream primer for amplifying the CD16A gene sequence in example 6 was optimized to obtain the same downstream primer CD16A-R1 as in example 1.

The electrophoresis results of examples 5 and 6 are shown in FIG. 4 (numbers (1) and (2) in FIG. 4 correspond to the primers of examples 5 and 6, respectively), and the electrophoresis pattern shows that the amplification specificity of the primer of example 6 after optimization is optimal.

Example 7

The procedure is as in example 1, with the main difference that the primers used are different. Wherein, the primer sequence of the CD16A-158F-F1 is as follows: 5' -agagcggctccttacttctgcagggggcv ₁ tt-3’(SEQ ID NO:3)，v ₁ Is a combination of three primers of c base, a base and g base;

the primer sequences of CD16A-158V-R1 are: 5' -tctctctgaagacattttactctcd ₁ cb ₁ ac-3’(SEQ ID NO:4)，b ₁ Is t base, d ₁ G base; b ₁ C is a base, d ₁ A is a base; b ₁ G base, d ₁ Is a combination of t bases three primers.

The electrophoresis result pattern of the PCR product of this example is substantially the same as that of example 1 and is not repeated. The embodiment can also realize amplification of a target band by adopting a plurality of primer combinations with different mismatched bases, which proves that the primers introducing different mismatched bases can rapidly judge the genotype of the rs396991 locus through one-time PCR reaction, and verifies the feasibility of successfully identifying the genotype of the rs396991 locus by introducing the primers with different mismatched bases.

Comparative example 1

The main difference in the method of example 1 is that the primer set for detecting the rs396991 site polymorphism is different (mismatched base is not introduced), as shown in Table 13 below:

TABLE 13

Component (concentration)	Primer sequences
		CD16A-158F-F’(10μM)	5’-acagcggctcctacttctgcagggggcttt-3’(SEQ ID NO:1)
CD16A-158V-R’(10μM)	5’-tctctgaagacacatttttactcccaac-3’(SEQ ID NO:2)

Because the primer of the table 13 has a mismatch of only one base at the 3' -end when the primer is incompletely complementary with the rs396991 locus, the incompletely complementary fragment can be amplified, a nonspecific strip appears, the genotype of the rs396991 locus is difficult to judge through a PCR result, and therefore the primer without the mismatch cannot judge the polymorphism of the rs396991 locus by adopting a one-step method.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (10)

1. A primer, which is characterized by comprising a first primer group for detecting the rs396991 locus polymorphism of the CD16A gene;

the first primer set includes:

CD16A-158F：5’-acagcggctcctacttctgcagggggcttt-3’(SEQ ID NO:1)，

CD16A-158V：5’-tctctgaagacacatttttactcccaac-3’(SEQ ID NO:2)，

in the CD16A-158F and the CD16A-158V, at least 1 mismatched base is introduced within 5 bases near the 3' end except for the first base.

2. The primer of claim 1, wherein the sites into which mismatched bases are introduced are not adjacent.

3. The primer of claim 1, wherein the first primer set comprises:

CD16A-158F：5’-acagcggctcctacttctgcagggggcv ₁ tt-3’(SEQ ID NO:3)，

CD16A-158V：5’-tctctgaagacacatttttactcd ₁ cb ₁ ac-3 '(SEQ ID NO: 4) and/or 5' -tctctctgaagacattttactctcccb ₂ ac-3’(SEQ ID NO:5)，

Wherein the v ₁ At least one of a, c or g; said b ₁ At least one of t, c or g; said d ₁ At least one of a, t or g; said b ₂ At least one of t, c or gA kind of module is assembled in the module and the module is assembled in the module.

4. The primer set according to claim 3, wherein the first primer set comprises:

CD16A-158F-F1：5’-acagcggctcctacttctgcagggggcCtt-3’(SEQ ID NO:6)，

CD16A-158V-R1:5'-gtctctgaagacacatttttactcTcGac-3' (SEQ ID NO: 7); and/or

CD16A-158V-R1’：5’-tctctgaagacacatttttactcccCac-3’(SEQ ID NO:8)。

5. The primer of any one of claims 1-4, wherein the primer further comprises a second primer pair that amplifies a CD16A gene sequence comprising the rs396991 locus, the second primer pair comprising a forward primer and a reverse primer; the distance between the forward primer and the rs396991 locus is not equal to the distance between the reverse primer and the rs396991 locus;

the second primer pair comprises:

CD16A-F1：5’-gagaataagctctggcgatac-3’(SEQ ID NO:9)，

CD16A-R1:5'-gcacctcggttacttcatctgta-3' (SEQ ID NO: 10); and/or the number of the groups of groups,

CD16A-F2：5’-gagaataagctctggcgatac-3’(SEQ ID NO:9)，

CD16A-R2:5'-ctttgatgtgaccttagggaaact-3' (SEQ ID NO: 11); and/or the number of the groups of groups,

CD16Ar-F3：5’-gccaccgtcaccttattcct-3’(SEQ ID NO:12)，

CD16A-R3:5'-ttgcaggttccacacacagg-3' (SEQ ID NO: 13); and/or the number of the groups of groups,

CD16A-F4：5’-ggtcttggtatgacttcagttc-3’(SEQ ID NO:14)，

CD16A-R4：5’-ttgcaggttccacacacagg-3’(SEQ ID NO:13)。

6. the primer of claim 5, wherein the second primer pair comprises:

CD16A-F1:5'-gagaataagctctggcgatac-3' (SEQ ID NO: 9) and

CD16A-R1：5’-gcacctcggttacttcatctgta-3’(SEQ ID NO:10)。

7. use of the primer of any one of claims 1-6 for detecting a CD16A gene rs396991 locus polymorphism.

8. A kit for detecting a CD16A gene rs396991 locus polymorphism, said kit comprising the primer of any one of claims 1-6.

9. A method for detecting a CD16A gene rs396991 locus polymorphism, comprising the steps of:

PCR is performed using the primer according to any one of claims 1 to 6, or the kit according to claim 7 or 8, and the rs396991 locus polymorphism of the CD16A gene is determined based on the PCR product.

10. The method according to claim 9, wherein the method comprises the steps of:

s1: preparing a sample to be tested;

s2: taking the sample to be detected as a template, and carrying out PCR reaction by adopting the primer or the kit to obtain a PCR product;

s3: and (3) carrying out electrophoresis on the PCR product, and judging the rs396991 locus polymorphism of the CD16A gene according to the electrophoresis band.

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