阅读说明：本技术 Kir2dl4基因分型试剂盒和分型方法 (KIR2DL4 genotyping kit and genotyping method ) 是由 郑仲征 杜可明 廖宽镇 贺青青 李岱阳 于 2021-02-22 设计创作，主要内容包括：本发明公开一种KIR2DL4基因分型试剂盒和分型方法,其中,本发明KIR2DL4基因分型试剂盒包括扩增引物一、扩增引物二、扩增引物三、扩增引物四、扩增引物五、扩增引物六、测序引物一、测序引物二、测序引物三、测序引物四、测序引物五、测序引物六、测序引物七、测序引物八、测序引物九、测序引物十、测序引物十一以及测序引物十二。本发明通过多种扩增引物对KIR2DL4基因进行PCR扩增,多种测序引物对扩增的PCR产物进行测序扩增,进而对测序扩增产物进行测序,发现其他方法不易鉴定的新等位基因型,实现对KIR2DL4基因的高分辨分型。(The invention discloses a KIR2DL4 genotyping kit and a genotyping method, wherein the KIR2DL4 genotyping kit comprises a first amplification primer, a second amplification primer, a third amplification primer, a fourth amplification primer, a fifth amplification primer, a sixth amplification primer, a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer. According to the invention, the KIR2DL4 gene is subjected to PCR amplification through multiple amplification primers, and the amplified PCR product is subjected to sequencing amplification through multiple sequencing primers, so that the sequencing amplification product is sequenced, a new allele type which is difficult to identify by other methods is found, and the high-resolution typing of the KIR2DL4 gene is realized.)

1. A KIR2DL4 genotyping kit, comprising:

the nucleotide sequence of the amplification primer I is shown as SEQ ID No. 1;

the nucleotide sequence of the amplification primer II is shown as SEQ ID No. 2;

the third amplification primer has a nucleotide sequence shown as SEQ ID No. 3;

the fourth amplification primer, the nucleotide sequence of which is shown as SEQ ID No. 4;

the amplification primer V has a nucleotide sequence shown as SEQ ID No. 5;

the sixth amplification primer, the nucleotide sequence of which is shown as SEQ ID No. 6;

the nucleotide sequence of the sequencing primer I is shown as SEQ ID No. 7;

a sequencing primer II, the nucleotide sequence of which is shown as SEQ ID No. 8;

a sequencing primer III, the nucleotide sequence of which is shown as SEQ ID No. 9;

a sequencing primer IV, wherein the nucleotide sequence is shown as SEQ ID No. 10;

a sequencing primer V, the nucleotide sequence of which is shown as SEQ ID No. 11;

a sequencing primer six, the nucleotide sequence of which is shown as SEQ ID No. 12;

a sequencing primer seven, the nucleotide sequence of which is shown as SEQ ID No. 13;

a sequencing primer eight, the nucleotide sequence of which is shown as SEQ ID No. 14;

a ninth sequencing primer, the nucleotide sequence of which is shown as SEQ ID No. 15;

a sequencing primer ten, the nucleotide sequence of which is shown as SEQ ID No. 16;

the eleven sequencing primers have nucleotide sequences shown as SEQ ID No. 17; and

and a twelve sequencing primer, wherein the nucleotide sequence is shown as SEQ ID No. 18.

2. A KIR2DL4 genotyping method is characterized by comprising the following steps:

(1) obtaining the genome DNA of a sample to be detected;

(2) performing PCR amplification by using the first amplification primer, the second amplification primer, the third amplification primer, the fourth amplification primer, the fifth amplification primer and the sixth amplification primer respectively by taking the genomic DNA as a template to obtain corresponding amplification products;

(3) amplifying the corresponding amplification products respectively by using a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer to obtain corresponding sequencing amplification products;

(4) sequencing the sequencing amplification product, analyzing SNP locus information in a nucleotide sequence, and comparing the SNP locus information with a public database to obtain the genotyping of KIR2DL 4;

wherein, the nucleotide sequence of the first amplification primer is shown as SEQ ID No.1, the nucleotide sequence of the second amplification primer is shown as SEQ ID No.2, the nucleotide sequence of the third amplification primer is shown as SEQ ID No.3, the nucleotide sequence of the fourth amplification primer is shown as SEQ ID No.4, the nucleotide sequence of the fifth amplification primer is shown as SEQ ID No.5, the nucleotide sequence of the sixth amplification primer is shown as SEQ ID No.6, the nucleotide sequence of the first sequencing primer is shown as SEQ ID No.7, the nucleotide sequence of the second sequencing primer is shown as SEQ ID No.8, the nucleotide sequence of the third sequencing primer is shown as SEQ ID No.9, the nucleotide sequence of the fourth sequencing primer is shown as SEQ ID No.10, the nucleotide sequence of the fifth sequencing primer is shown as SEQ ID No.11, and the nucleotide sequence of the sixth sequencing primer is shown as SEQ ID No.12, the nucleotide sequence of the sequencing primer seven is shown as SEQ ID No.13, the nucleotide sequence of the sequencing primer eight is shown as SEQ ID No.14, the nucleotide sequence of the sequencing primer nine is shown as SEQ ID No.15, the nucleotide sequence of the sequencing primer ten is shown as SEQ ID No.16, the nucleotide sequence of the sequencing primer eleven is shown as SEQ ID No.17, and the nucleotide sequence of the sequencing primer twelve is shown as SEQ ID No. 18.

Technical Field

The invention relates to the technical field of biology, in particular to a KIR2DL4 genotyping kit and a genotyping method.

Background

Natural Killer cells (NK cells) play an important role in innate and adaptive immunity by acting as cytokines and cytotoxic agents in the human body to combat infection and kill malignant cells. Killer cell Immunoglobulin-like receptors (kirers) are a group of Immunoglobulin-like superfamily members that are mainly expressed on the surface of NK cells and partial T cells, and are capable of specifically recognizing Human major histocompatibility complex MHC-class I molecules (Human leukcyte Antigen I, HLA-I), mediating and regulating the killing function of NK cells and partial effector T cells. An increasing number of clinical transplantation and animal experimental evidence suggests that NK cell allogenic responses triggered by KIR incompatibility in donor and recipient are beneficial for the prognosis of allogeneic hematopoietic stem cell transplantation and organ transplantation. The heterogenous reactive NK cells can specifically kill Host antigen presenting cells and leukemia cells, so that the risk of Graft-Versus-Host Disease (GVHD) is reduced, rejection is relieved, and the effect of Graft-Versus-leukemia is achieved. In addition, KIR has relevance to numerous diseases, including autoimmune diseases, viral infection diseases, tumors, pregnancy-related diseases, and the like.

The KIR gene is located on chromosome 19q13.4 and has high polymorphism, and 14 KIR functional genes and 2 pseudogenes are detected at present. By 12 months 2019, 1110 alleles have been found. The killer cell immunoglobulin-like receptor KIR2DL4 gene is a member of the KIR gene family, and has 8 exons (exons 1, 2, 3, 5, 6, 7, 8, and 9) in total, with exon 4 deleted.

With the development of transplantation immunity research, the scheme of HLA semi-compatible transplantation is popularized, and how to choose the optimal donor from a plurality of donors becomes a difficult problem for clinical experts. Under the situation, KIR genotyping is paid more and more attention by clinical experts, and the continuous development of detection technology also leads to the trend of high-resolution genotyping. KIR typing methods include sequence-specific primer PCR (PCR-SSP), reverse transcription PCR (RT-PCR), PCR-sequence-specific oligonucleotide probe (PCR-SSO), and the like. However, the above-mentioned various detection methods cannot achieve high-resolution typing of KIR2DL4 gene.

The above is only for the purpose of assisting understanding of the technical solutions of the present invention, and does not represent an admission that the above is the prior art.

Disclosure of Invention

The invention mainly aims to provide a KIR2DL4 genotyping kit, aiming at realizing high-resolution genotyping of KIR2DL4 genes.

In order to achieve the above object, the present invention provides, in a first aspect, a KIR2DL4 genotyping kit, comprising:

the nucleotide sequence of the amplification primer I is shown as SEQ ID No. 1;

the nucleotide sequence of the amplification primer II is shown as SEQ ID No. 2;

the third amplification primer has a nucleotide sequence shown as SEQ ID No. 3;

the fourth amplification primer, the nucleotide sequence of which is shown as SEQ ID No. 4;

the amplification primer V has a nucleotide sequence shown as SEQ ID No. 5;

the sixth amplification primer, the nucleotide sequence of which is shown as SEQ ID No. 6;

the nucleotide sequence of the sequencing primer I is shown as SEQ ID No. 7;

a sequencing primer II, the nucleotide sequence of which is shown as SEQ ID No. 8;

a sequencing primer III, the nucleotide sequence of which is shown as SEQ ID No. 9;

a sequencing primer IV, wherein the nucleotide sequence is shown as SEQ ID No. 10;

a sequencing primer V, the nucleotide sequence of which is shown as SEQ ID No. 11;

a sequencing primer six, the nucleotide sequence of which is shown as SEQ ID No. 12;

a sequencing primer seven, the nucleotide sequence of which is shown as SEQ ID No. 13;

a sequencing primer eight, the nucleotide sequence of which is shown as SEQ ID No. 14;

a ninth sequencing primer, the nucleotide sequence of which is shown as SEQ ID No. 15;

a sequencing primer ten, the nucleotide sequence of which is shown as SEQ ID No. 16;

the eleven sequencing primers have nucleotide sequences shown as SEQ ID No. 17; and

and a twelve sequencing primer, wherein the nucleotide sequence is shown as SEQ ID No. 18.

In a second aspect, the invention provides a KIR2DL4 genotyping method, which comprises the following steps:

(1) obtaining the genome DNA of a sample to be detected;

(2) performing PCR amplification by using the first amplification primer, the second amplification primer, the third amplification primer, the fourth amplification primer, the fifth amplification primer and the sixth amplification primer respectively by taking the genomic DNA as a template to obtain corresponding amplification products;

(3) amplifying the corresponding amplification products respectively by using a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer to obtain corresponding sequencing amplification products;

(4) sequencing the sequencing amplification product, analyzing SNP locus information in a nucleotide sequence, and comparing the SNP locus information with a public database to obtain the genotyping of KIR2DL 4;

wherein, the nucleotide sequence of the first amplification primer is shown as SEQ ID No.1, the nucleotide sequence of the second amplification primer is shown as SEQ ID No.2, the nucleotide sequence of the third amplification primer is shown as SEQ ID No.3, the nucleotide sequence of the fourth amplification primer is shown as SEQ ID No.4, the nucleotide sequence of the fifth amplification primer is shown as SEQ ID No.5, the nucleotide sequence of the sixth amplification primer is shown as SEQ ID No.6, the nucleotide sequence of the first sequencing primer is shown as SEQ ID No.7, the nucleotide sequence of the second sequencing primer is shown as SEQ ID No.8, the nucleotide sequence of the third sequencing primer is shown as SEQ ID No.9, the nucleotide sequence of the fourth sequencing primer is shown as SEQ ID No.10, the nucleotide sequence of the fifth sequencing primer is shown as SEQ ID No.11, and the nucleotide sequence of the sixth sequencing primer is shown as SEQ ID No.12, the nucleotide sequence of the sequencing primer seven is shown as SEQ ID No.13, the nucleotide sequence of the sequencing primer eight is shown as SEQ ID No.14, the nucleotide sequence of the sequencing primer nine is shown as SEQ ID No.15, the nucleotide sequence of the sequencing primer ten is shown as SEQ ID No.16, the nucleotide sequence of the sequencing primer eleven is shown as SEQ ID No.17, and the nucleotide sequence of the sequencing primer twelve is shown as SEQ ID No. 18.

The KIR2DL4 genotyping kit comprises a first amplification primer, a second amplification primer, a third amplification primer, a fourth amplification primer, a fifth amplification primer, a sixth amplification primer, a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer; the KIR2DL4 gene is subjected to PCR amplification through multiple amplification primers, and the amplified PCR product is subjected to sequencing amplification through multiple sequencing primers, so that the sequencing amplification product is sequenced, a new allele type which is difficult to identify by other methods is found, and high-resolution typing of the KIR2DL4 gene is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.

FIG. 1 is an agarose gel electrophoresis of the PCR amplification product of the first embodiment;

FIG. 2 is a diagram showing the result of the 1524 th SNP alignment of example sample;

FIG. 3 is a diagram showing the alignment result of the 2651 th SNP of example II;

FIG. 4 is a diagram showing the result of the 2864 th SNP alignment of example sample II;

FIG. 5 is a diagram showing the result of the comparison of the 9838 th SNP in example II;

FIG. 6 is a diagram showing the result of the 10659 th SNP alignment of example II.

The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.

Detailed Description

It should be noted that if the description of "first", "second", etc. is provided in the embodiment of the present invention, the description of "first", "second", etc. is only for descriptive purposes and is not to be construed as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout is to include three juxtapositions, exemplified by "A and/or B" including either scheme A, or scheme B, or a scheme in which both A and B are satisfied.

The invention provides a KIR2DL4 genotyping kit.

Referring to tables 1 and 2, the KIR2DL4 genotyping kit provided by the invention comprises an amplification primer i, an amplification primer ii, an amplification primer iii, an amplification primer iv, a sequencing primer i, a sequencing primer ii, a sequencing primer iii, a sequencing primer iv, and a sequencing primer iv; wherein, the nucleotide sequence of the first amplification primer is shown as SEQ ID No.1, the nucleotide sequence of the second amplification primer is shown as SEQ ID No.2, the nucleotide sequence of the third amplification primer is shown as SEQ ID No.3, the nucleotide sequence of the fourth amplification primer is shown as SEQ ID No.4, the nucleotide sequence of the fifth amplification primer is shown as SEQ ID No.5, the nucleotide sequence of the sixth amplification primer is shown as SEQ ID No.6, the nucleotide sequence of the first sequencing primer is shown as SEQ ID No.7, the nucleotide sequence of the second sequencing primer is shown as SEQ ID No.8, the nucleotide sequence of the third sequencing primer is shown as SEQ ID No.9, the nucleotide sequence of the fourth sequencing primer is shown as SEQ ID No.10, the nucleotide sequence of the fifth sequencing primer is shown as SEQ ID No.11, and the nucleotide sequence of the sixth sequencing primer is shown as SEQ ID No.12, the nucleotide sequence of the sequencing primer seven is shown as SEQ ID No.13, the nucleotide sequence of the sequencing primer eight is shown as SEQ ID No.14, the nucleotide sequence of the sequencing primer nine is shown as SEQ ID No.15, the nucleotide sequence of the sequencing primer ten is shown as SEQ ID No.16, the nucleotide sequence of the sequencing primer eleven is shown as SEQ ID No.17, and the nucleotide sequence of the sequencing primer twelve is shown as SEQ ID No. 18.

Referring to table 1, the first amplification primer is an amplification upstream primer, the second amplification primer is an amplification downstream primer, and the first amplification primer and the second amplification primer specifically amplify a sequence of a 5' UTR region, a 1 st exon, a 1 st intron, a 2 nd exon, a 2 nd intron, a 3 rd exon (pseudo exon), a 3 rd intron, a 4 th exon, a 4 th intron, a 5 th exon and a part of a 5 th intron of a KIR2DL4 gene, which is named as P1; the third amplification primer is an amplification upstream primer, the fourth amplification primer is an amplification downstream primer, and the third amplification primer and the fourth amplification primer specifically amplify the sequences of a part of the 5 th intron, a part of the 6 th exon and a part of the 6 th intron of the KIR2DL4 gene, wherein the sequence is named as P2; the fifth amplification primer is an amplification upstream primer, the sixth amplification primer is an amplification downstream primer, and the fifth amplification primer and the sixth amplification primer specifically amplify sequences of a part of 6 th intron, 7 th exon, 7 th intron, 8 th exon, 8 th intron, 9 th exon and a part of 3' UTR region of the KIR2DL4 gene, wherein the sequence is named as P3.

The 2DL4 x 0010201 gene from KIR public database (https:// www.ebi.ac.uk/ipd/KIR /) was selected as a control gene and the position of each primer in the genome was aligned. The total length of the control gene covers from 5 'UTR region to 3' UTR region of KIR2DL4 gene, total 11177 bp.

TABLE 1 PCR amplification primer information

The sequencing primer I, the sequencing primer II, the sequencing primer III, the sequencing primer IV, the sequencing primer V, the sequencing primer VI, the sequencing primer VII, the sequencing primer eight, the sequencing primer nine, the sequencing primer eleven and the sequencing primer twelve are used for sequencing and amplifying the purified PCR amplification product.

Referring to table 2, the first sequencing primer is an amplification upstream primer, the second sequencing primer is an amplification downstream primer, and the first sequencing primer and the second sequencing primer pair are used for sequencing and amplifying the 1 st exon and the 2 nd exon sequences in P1; the third sequencing primer is an amplification upstream primer, the fourth sequencing primer is an amplification downstream primer, and the third sequencing primer and the fourth sequencing primer perform sequencing amplification on the 3 rd exon sequence in the P1; the sequencing primer five is an amplification upstream primer, the sequencing primer six is an amplification downstream primer, and the sequencing primer five and the sequencing primer six perform sequencing amplification on the 5 th exon sequence in the P1; the seventh sequencing primer is an amplification upstream primer, the eighth sequencing primer is an amplification downstream primer, and the seventh sequencing primer and the eighth sequencing primer perform sequencing amplification on the 6 th exon sequence in the P2; the sequencing primer nine is an amplification upstream primer, the sequencing primer ten is an amplification downstream primer, and the sequencing primer nine and the sequencing primer ten carry out sequencing amplification on the 7 th exon sequence in the P3; the sequencing primer eleven is an amplification upstream primer, the sequencing primer twelve is an amplification downstream primer, and sequencing amplification is performed on the 8 th exon and the 9 th exon in the twelve pairs of P3 of the sequencing primer eleven and the sequencing primer eleven.

Since exon 4 of KIR2DL4 gene is a deleted exon, the sequencing primer is not designed for exon 4 for sequencing amplification in the invention.

TABLE 2 sequencing primer information

The invention also provides a KIR2DL4 genotyping method, which comprises the following steps:

(1) obtaining the genome DNA of a sample to be detected; (2) performing PCR amplification by using the first amplification primer, the second amplification primer, the third amplification primer, the fourth amplification primer, the fifth amplification primer and the sixth amplification primer respectively by taking the genomic DNA as a template to obtain corresponding amplification products; (3) amplifying the corresponding amplification products respectively by using a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer to obtain corresponding sequencing amplification products; (4) sequencing the sequencing amplification product, analyzing SNP locus information in a nucleotide sequence, and comparing the SNP locus information with a public database to obtain the genotyping of KIR2DL 4; wherein, the nucleotide sequence of the first amplification primer is shown as SEQ ID No.1, the nucleotide sequence of the second amplification primer is shown as SEQ ID No.2, the nucleotide sequence of the third amplification primer is shown as SEQ ID No.3, the nucleotide sequence of the fourth amplification primer is shown as SEQ ID No.4, the nucleotide sequence of the fifth amplification primer is shown as SEQ ID No.5, the nucleotide sequence of the sixth amplification primer is shown as SEQ ID No.6, the nucleotide sequence of the first sequencing primer is shown as SEQ ID No.7, the nucleotide sequence of the second sequencing primer is shown as SEQ ID No.8, the nucleotide sequence of the third sequencing primer is shown as SEQ ID No.9, the nucleotide sequence of the fourth sequencing primer is shown as SEQ ID No.10, the nucleotide sequence of the fifth sequencing primer is shown as SEQ ID No.11, and the nucleotide sequence of the sixth sequencing primer is shown as SEQ ID No.12, the nucleotide sequence of the sequencing primer seven is shown as SEQ ID No.13, the nucleotide sequence of the sequencing primer eight is shown as SEQ ID No.14, the nucleotide sequence of the sequencing primer nine is shown as SEQ ID No.15, the nucleotide sequence of the sequencing primer ten is shown as SEQ ID No.16, the nucleotide sequence of the sequencing primer eleven is shown as SEQ ID No.17, and the nucleotide sequence of the sequencing primer twelve is shown as SEQ ID No. 18.

The method comprises the following specific steps:

1) PCR amplification

For the amplification primer information, see table 1, for the reaction system, see table 3, and for the reaction conditions, see table 4.

TABLE 3 PCR reaction System

TABLE 4 PCR reaction procedure

2) PCR product purification

After the PCR amplification is finished, the amplification product is purified, and the interference of the PCR reaction reagent on the subsequent sequencing reaction is avoided. The reaction system for purification of the amplification product is shown in Table 5, and the purification conditions are shown in Table 6.

TABLE 5 amplification product purification System

Reagent	Volume of
		Takara Alkaline Phosphatase	0.5μl
Takara Exonuclease I	0.5μl
		Amplification product	25.0μl

TABLE 6 amplification product purification conditions

Step (ii) of	Temperature of	Time
			1	37℃	90min
2	80℃	15min

3) Sequencing amplification

And sequencing and amplifying the purified amplification product. For information on sequencing amplification primers, see table 2, for reaction system, see table 7, and for reaction conditions, see table 8.

TABLE 7 sequencing amplification reaction System

Reagent	Volume of
		BigDye Terminator v3.1	0.5μl
5×BigDye Sequencing Buffer	0.75μl
		Sequencing primer (3.2. mu.M)	1.0μl
Purified PCR amplification product	1.5μl
		ddH2O	Adding to a total volume of 5.0. mu.l

TABLE 8 sequencing amplification reaction procedure

4) Sequencing amplification product purification

After the sequencing amplification reaction is completed, purifying the sequencing amplification product so as to prevent the sequencing amplification reagent from interfering the sequencing process, and the steps are as follows:

a) reacting absolute ethyl alcohol with ddH₂Mixing O, and preparing 85% ethanol and 70% ethanol;

b) after the sequencing reaction plate is centrifuged, 2 mu l of EDTA solution and 40 mu l of 85% ethanol are respectively added into each reaction hole, a silica gel pad is covered on each reaction hole, the mixture is fully vibrated for 3min, and the mixture is centrifuged at 4 ℃ and 3,000 Xg for 30 min;

c) taking out the sequencing reaction plate after the centrifugation is finished, spreading absorbent paper with enough thickness in a centrifugation hanging basket, slightly uncovering a silica gel pad of the sequencing reaction plate, inversely placing the sequencing reaction plate on the absorbent paper, and centrifuging the sequencing reaction plate and the absorbent paper together until the centrifugal force reaches 185 Xg;

d) adding 70% ethanol 50 μ l into each well, covering with silica gel pad, shaking for 2min, centrifuging at 4 deg.C and 3,000 Xg for 15 min;

e) repeating the step c;

f) drying the sequencing reaction plate for 20min in the dark, adding 10 mul of ultrapure formamide into each hole, covering a silica gel pad, performing instantaneous centrifugation, and reacting for 2min at 96 ℃ in a PCR instrument;

g) sequencing the purified sequencing reaction product by using an ABI3730xl sequencer;

h) and (3) comparing a sequencing sequence file generated by a sequencer with a 2DL4 x 0010201 reference sequence by using SnapGene software (https:// www.snapgene.com), generating corresponding SNP and position information thereof, and comparing with a KIR public database to obtain a high-resolution type of the KIR2DL4 gene.

The KIR2DL4 genotyping kit comprises a first amplification primer, a second amplification primer, a third amplification primer, a fourth amplification primer, a fifth amplification primer, a sixth amplification primer, a first sequencing primer, a second sequencing primer, a third sequencing primer, a fourth sequencing primer, a fifth sequencing primer, a sixth sequencing primer, a seventh sequencing primer, an eighth sequencing primer, a ninth sequencing primer, a tenth sequencing primer, an eleventh sequencing primer and a twelfth sequencing primer; the KIR2DL4 gene is subjected to PCR amplification through multiple amplification primers, and the amplified PCR product is subjected to sequencing amplification through multiple sequencing primers, so that the sequencing amplification product is sequenced, a new allele type which is difficult to identify by other methods is found, and high-resolution typing of the KIR2DL4 gene is realized.

Example one

Two positive samples subjected to KIR2DL4 gene low-resolution genotyping are randomly selected, and the kit and the genotyping method are adopted to perform KIR2DL4 gene genotyping on the samples so as to verify the genotyping effect of the invention. Extracting the genome DNA of the sample, and carrying out PCR amplification on the genome DNA. The results of PCR amplification are shown in FIG. 1. In FIG. 1, the bands of the agarose gel electrophoresis image are, from left to right, DL2000 DNA Marker, P1 sequence of sample (i), P1 sequence of sample (ii), P2 sequence of sample (i), P2 sequence of sample (ii), P3 sequence of sample (i) and P3 sequence of sample (ii). The sequence length of P1 is 2789bp, the sequence length of P2 is 401bp, the sequence length of P3 is 1160bp, and the result in the figure is consistent with the reality, which indicates that the sequence is successfully amplified.

Sequencing the purified sequencing amplification product to obtain 12 sequencing peak maps. The 12 sequencing peaks were each introduced into SnapGene software, and aligned With the Sequence of 2DL4 × 0010201 using the "Align With a Sequence Trace" function of the SnapGene software.

The alignment results show that no SNP is found in sample (i) relative to the reference sequence 2DL4 × 0010201, i.e. sample (i) is completely identical to reference sequence 2DL4 × 0010201 in exons 1, 2, 3, 5, 6, 7, 8 and 9.

Referring to fig. 2 to 6, the alignment results show that sample (c) has five SNPs in total relative to the reference sequence 2DL4 × 0010201. The first SNP is located at the 1524 th position of the 3 rd exon, the base of the sample (II) at the position is R (marked by a black frame), namely the base of the sample (II) at the position is A or G, and the base of the 2DL 4-0010201 reference sequence at the position is A. The second SNP is located at 2651 th of exon 5, the base of sample (ii) at this position is R (marked by a black box), that is, the base of sample (ii) at this position is A or G, and the base of 2DL4 × 0010201 reference sequence at this position is A. The third SNP is positioned at 2864 th of the 5 th exon, the base of the sample (II) at the position is S (marked by a black frame), namely the base of the sample (II) at the position is C or G, and the base of the 2DL 4-0010201 reference sequence at the position is G. The fourth SNP is located at the 9838 th site of the 7 th exon, the base of the sample (II) at the site is Y (marked by a black box), namely the base of the sample (II) at the site is C or T, and the base of the 2DL 4-0010201 reference sequence at the site is C. The fifth SNP is located at 10659 th of the 9 th exon, the base of the sample (II) at the position is R (marked by a black box), namely the base of the sample (II) at the position is A or G, and the base of the 2DL 4-0010201 reference sequence at the position is A. Sample 2, 6, and 8 th exons were completely identical in sequence to 2DL4 × 0010201, and no other SNPs were found.

The public database of KIR is queried, and the SNP result obtained by the comparison of the sample in this embodiment is consistent with the reference sequence, and the high-resolution typing result of the sample (KIR 2DL 4) is 2DL4 × 00102 homozygote. The SNP result obtained by the comparison of the sample (II) is consistent with heterozygotes of 2DL 4X 00102 and 2DL 4X 011, and the high-resolution typing result of the sample (II) KIR2DL4 is 2DL 4X 00102 and 2DL 4X 011. The above results demonstrate that the kit and typing method of the present invention can achieve high resolution typing of KIR2DL 4.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen silvergrass Beiken precision medicine Limited

SHANGHAI TISSUEBANK MEDICAL INSPECTION Co.,Ltd.

SHANGHAI TISSUEBANK BIOTECHNOLOGY Co.,Ltd.

Shanghai Yushuo Beiken Gene Technology Co., Ltd.

<120> KIR2DL4 genotyping kit and genotyping method

<130>

<160> 18

<170> PatentIn version 3.3

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<212> DNA

<213> Artificial Synthesis

<400> 15

atgagagaca atccaca 17

<210> 16

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 16

aacacacacc tgcgtgct 18

<210> 17

<211> 15

<212> DNA

<213> Artificial Synthesis

<400> 17

ttggtatctg ttcat 15

<210> 18

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 18

gaggaagagt gatgctctaa gatgg 25