CN112746100A - Primer, kit and method for KIR genotyping - Google Patents
Primer, kit and method for KIR genotyping Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a primer combination for KIR gene typing, a kit containing the primer combination and a method for typing KIR genes by adopting the primer combination or the kit. By adopting the primer combination of the invention, all sites of the KIR gene can be amplified by using 16 amplification tubes, and all types which can be distinguished by the exon sequences can be detected under the condition that the exon reference sequences are complete.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to primers, a kit and a method for KIR genotyping.
Background
Organ transplantation is the transplantation of a healthy organ into a recipient to replace an organ that has lost its function due to a fatal disease and to restore its function rapidly. The bone marrow transplantation is to transfuse normal bone marrow into patient's body via vein to replace pathological bone marrow and to rebuild the hemopoietic function and immunity of patient, so as to treat various blood diseases including leukemia. In bone marrow or organ transplantation, rejection has been a major factor affecting graft survival. It has long been generally accepted by the medical community that Human Leukocyte Antigen (HLA) is the human leukocyte antigen that is crucial for the recognition of foreign antigens in rejection. However, with the progress of research, patients who find that part of HLA genotypes are completely matched still have the phenomenon of graft rejection. Therefore, in addition to HLA-concordance or noncompliance, the family of natural killer cell (NK) surface killer immunoglobulin-like receptors (KIRs) is of increasing interest.
There are currently 17 known members of the KIR receptor family, divided into activating receptor type S and inhibitory receptor type L, KIR2DL1-4, KIR2DL5A, KIR2DL5B, KIR2DS1-5, KIR3DL1-3, KIR3DS1, and pseudogenes KIR2DP1 and KIR3DP 1. The KIR gene family has haplotype difference in gene number and allelic polymorphism among individual genes. The diversity of KIR genes is mainly expressed as: (1) the number of KIR genes is different among different NK cell clones and different individuals, and the different individuals can show different haplotypes; (2) there are many different alleles of the same KIR locus, most of which are non-synonymous mutations. Thus, the diversity of KIR gene composition and allelic polymorphism make it likely that KIR typing will be different in donors and recipients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) even when HLA class I and class II sites are all in agreement. Human KIR haplotypes share two types: type a and type B, whose composition and distribution vary widely between different races and individuals, the B haplotype contains different combinations of a variety of B-characteristic genes (KIR2DL2, 2DL5, 2DS1, 2DS2, 2DS3, 2DS5 or 3DS 1). The polymorphism of the haplotype A is mainly embodied as allelic polymorphism; while haplotype B is mainly represented by polymorphisms in gene constitution, which have large differences in gene types and numbers among different individuals and relatively small differences in allele levels.
Research has shown that KIR genotypes and haplotypes play a role in allo-HSCT. For the Han population in China, selection of donors with haplotype B (A/B or B/B, collectively referred to as B/x) helps to increase the overall survival rate (OS) after allogeneic hematopoietic stem cell transplantation.
KIR receptors are expressed on NK cells and T cells, and the ligands are MHC-I molecules. Their interactions play an important role in both innate and adaptive immunity. KIR and MHC class I molecules are encoded by unrelated polymorphic gene families. Various combinations of KIR and MHC class I molecules can affect the body's anti-infective immunity, autoimmune disease and various pregnancy complications and the prognosis of transplantation of sculpted stem cells. These correlations suggest that polymorphisms in KIR and MHC-1 molecules may play an important role in promoting human survival in epidemic infections and affecting human reproduction and population growth during human evolution.
Therefore, the KIR type of the donor needs to be determined before bone marrow or organ transplantation, so that the risk of rejection of the potential donor by the recipient can be evaluated, and the donor with high rejection risk can be avoided under the condition of considering the HLA type.
However, the existing method for performing KIR typing by Sequencing Based Typing (SBT) is high in cost, cumbersome in operation and long in time. Therefore, a rapid, accurate and cost-effective method for typing KIR genes is needed.
Disclosure of Invention
The invention provides a complete set of primers, a kit and a method for KIR gene typing, aiming at the urgent need of a rapid, accurate and cost-effective KIR gene typing method in clinic. The typing method is simple, convenient and accurate, and has wide coverage types.
To this end, the invention provides a primer combination for KIR genotyping, which consists of primers shown in SEQ ID NO. 1-39.
In a preferred embodiment of the invention, the primer combination consists of the primers of groups 1 to 16, wherein group 1 consists of the primers shown in SEQ ID Nos. 1 to 3, group 2 consists of the primers shown in SEQ ID Nos. 4 to 5, group 3 consists of the primers shown in SEQ ID Nos. 6 to 7, group 4 consists of the primers shown in SEQ ID Nos. 8 to 11, group 5 consists of the primers shown in SEQ ID Nos. 12 to 13, group 6 consists of the primers shown in SEQ ID Nos. 14 to 15, group 7 consists of the primers shown in SEQ ID Nos. 16 to 17, group 8 consists of the primers shown in SEQ ID Nos. 18 to 19, group 9 consists of the primers shown in SEQ ID Nos. 20 to 22, group 10 consists of the primers shown in SEQ ID Nos. 23 to 24, group 11 consists of the primers shown in SEQ ID Nos. 25 to 26, group 12 consists of primers shown by SEQ ID NO.27-28, group 13 consists of primers shown by SEQ ID NO.29-30, group 14 consists of primers shown by SEQ ID NO.31-33, group 15 consists of primers shown by SEQ ID NO.34-37, and group 16 consists of primers shown by SEQ ID NO. 38-39.
In a preferred embodiment of the present invention, a working solution is separately prepared for each set of primer combination, wherein the concentration of the primer of group 1 is 2.5 pmol/. mu.L, the concentration of the primer of group 2 is 1 pmol/. mu.L, the concentration of the primer of group 3 is 2 pmol/. mu.L, the concentration of the primer of group 4 is 2 pmol/. mu.L, the concentration of the primer of group 5 is 2.5 pmol/. mu.L, the concentration of the primer of group 6 is 1 pmol/. mu.L, the concentration of the primer of group 7 is 4 pmol/. mu.L, the concentration of the primer of group 8 is 3 pmol/. mu.L, the concentration of the primer of group 9 is 1 pmol/. mu.L, the concentration of the primer of group 10 is 2 pmol/. mu.L, the concentration of the primer of group 11 is 3 pmol/. mu.L, the concentration of the primer of group 12 is 2.5 pmol/. mu.L, the concentration of the primer of group 13 is 12 pmol/. mu.L, the concentration of the primer of, the concentration of the primer in group 15 was 1 pmol/. mu.L, and the concentration of the primer in group 16 was 0.4 pmol/. mu.L.
The primers for KIR genotyping of the present invention and their concentrations in the PCR reaction solution are shown in Table 1.
TABLE 1 primers for KIR genotyping and their concentrations in PCR reactions
In another aspect, the invention provides a kit for KIR genotyping, which comprises the primer combination disclosed by the invention.
In another aspect, the invention provides an application of the primer combination in preparation of a kit for KIR genotyping.
In another aspect, the invention provides a primer combination or an application of the kit in KIR genotyping.
In a final aspect of the invention, there is provided a method for genotyping KIR, comprising the steps of:
1. obtaining DNA of a sample to be detected;
2. carrying out PCR amplification by using the primer combination or the kit of the invention and the DNA obtained in the step 1 as a template;
3. and judging the KIR genotype of the sample to be detected according to the amplification result.
In a preferred embodiment of the present invention, a working solution is separately prepared for each set of primer combination, wherein the concentration of the primer of group 1 is 2.5 pmol/. mu.L, the concentration of the primer of group 2 is 1 pmol/. mu.L, the concentration of the primer of group 3 is 2 pmol/. mu.L, the concentration of the primer of group 4 is 2 pmol/. mu.L, the concentration of the primer of group 5 is 2.5 pmol/. mu.L, the concentration of the primer of group 6 is 1 pmol/. mu.L, the concentration of the primer of group 7 is 4 pmol/. mu.L, the concentration of the primer of group 8 is 3 pmol/. mu.L, the concentration of the primer of group 9 is 1 pmol/. mu.L, the concentration of the primer of group 10 is 2 pmol/. mu.L, the concentration of the primer of group 11 is 3 pmol/. mu.L, the concentration of the primer of group 12 is 2.5 pmol/. mu.L, the concentration of the primer of group 13 is 12 pmol/. mu.L, the concentration of the primer of, the concentration of the primer in group 15 was 1 pmol/. mu.L, and the concentration of the primer in group 16 was 0.4 pmol/. mu.L.
In a preferred embodiment of the invention, the PCR amplification system is:
mu.L of PCR buffer, 1. mu.L of 2.5mM dNTPs, 0.4. mu.L of control, 1. mu.L of primer combination, 1. mu.L of cresol red, 1. mu.L of sample DNA at 10-30 ng/. mu.L, 0.07. mu.L of Taq enzyme, and 10. mu.L of DNase-Free ddH2O was used to complement the reaction system.
In a preferred embodiment of the invention, the PCR amplification conditions are:
96 ℃ for 2 minutes, followed by 10 cycles of 94 ℃ for 10 seconds and 65 ℃ for 60 seconds, followed by 20 cycles of 94 ℃ for 10 seconds, 61 ℃ for 50 seconds, 72 ℃ for 30 seconds, and 15 ℃ in this order.
As apparent from the above description, the present invention has the following advantages as compared with the prior art.
1. Simplicity: aiming at a single sample, the amplification primer combination is divided into 16 amplification tubes to amplify all sites of the KIR gene. The method is simple and convenient to operate, short in time consumption and low in experiment cost, and the requirement for sample parting can be met only by trace detection of materials.
2. The accuracy is as follows: by utilizing the primer combination and the method, the typing detection is carried out on 6 KIR standards (UCLAKIRPanceSet, UCLAImmunogenetics center) covering common types, and the typing result is completely correct. In addition, 140 known KIR typing results are subjected to typing detection, and the typing results completely conform to each other.
3. The coverage type is wide: with the primer combinations and methods described herein, all types that can be distinguished by exon sequences can be detected with complete exon reference sequences.
In conclusion, the KIR genotyping method is simple, convenient and accurate and has wide coverage type.
Drawings
FIG. 1: DNA banding patterns obtained by the combined amplification of the 16 pairs of primers of the invention.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, but not limiting, of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall into the protection scope of the present invention.
Example 1: design and Synthesis of primers
In this example, the KIR exon sequences required for PCR primer design are derived from KIR Data base, website:https://www.ebi.ac.uk/ipd/kir/。
primer Premier 6.0 software is adopted for Primer design, and the designed primers are compared in a KIR database to confirm that the group of primers can specifically amplify the required fragments.
The primers were synthesized by Shanghai bioengineering technology, Inc., and the specific sequences are shown in Table 1.
Example 2: screening of amplification primers
And randomly selecting 96 DNA samples (containing a negative control), wherein all the samples are from clinical scientific research service samples of Shanghai Shuoge Beiken gene science and technology limited, amplifying the KIR gene exons, and judging the amplification efficiency and specificity of the primer group by agarose gel electrophoresis.
1. DNA extraction
DNA extraction was performed according to the QIAampDNAodoBloodMinikit kit, followed by DNase-Free ddH2Diluting O to 10-30 ng/. mu.L for standby.
2. PCR amplification system
10 μ L of amplification system was used: mu.L of 2 GC Buffer I (stored at 4 ℃) (TAKARA, cat # RR02 AG); 1 μ L of 2.5mM dNTPs (TakaraBi)o); 0.4. mu.L of Control (2.5 p); 1. mu.L of a primer set (primers were synthesized from Shanghai); 1 μ L cresol red (1mg/ml, 4 ℃ storage); 1 μ L of 10-30 ng/. mu.L sample DNA; 0.07. mu.L of La Taq enzyme (-20 ℃ C.) (TAKARA, cat # RR02 MA); with DNase-Free ddH2And O is used for complementing the reaction system.
3. PCR reaction conditions
The following PCR reaction conditions were used:
4. electrophoretic detection and primer screening
And (3) carrying out 1.5% agarose electrophoresis on the PCR product for 150v, detecting after 30min, and judging the amplification efficiency and specificity of the primer combination according to a band obtained by electrophoresis. And finally determining the 16 groups of amplification primers through detection analysis.
5. Analysis of results
And after obtaining the gel image, judging the negative and positive of the corresponding site according to the sizes of the specific bands corresponding to different amplification primer combinations to obtain the KIR genotype of the detection sample.
As shown in the attached figure 1, when the primer combination of the invention is used for detecting a clinical research service sample from Shanghai Shuo Beiken gene technology Co., Ltd, under the condition that a control band is ensured to appear, specific bands are amplified at the other 15 sites except for the 2DL3 site, which indicates that the 2DL3 site of the sample is negative, and the other 15 sites are positive.
Sequence listing
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Claims (10)
1. A primer combination for KIR genotyping, which consists of primers shown in SEQ ID NO. 1-39.
2. The primer combination according to claim 1, which further comprises primers of groups 1 to 16, wherein group 1 comprises primers shown by SEQ ID Nos. 1 to 3, group 2 comprises primers shown by SEQ ID Nos. 4 to 5, group 3 comprises primers shown by SEQ ID Nos. 6 to 7, group 4 comprises primers shown by SEQ ID Nos. 8 to 11, group 5 comprises primers shown by SEQ ID Nos. 12 to 13, group 6 comprises primers shown by SEQ ID Nos. 14 to 15, group 7 comprises primers shown by SEQ ID Nos. 16 to 17, group 8 comprises primers shown by SEQ ID Nos. 18 to 19, group 9 comprises primers shown by SEQ ID Nos. 20 to 22, group 10 comprises primers shown by SEQ ID Nos. 23 to 24, group 11 comprises primers shown by SEQ ID Nos. 25 to 26, group 12 consists of primers shown by SEQ ID NO.27-28, group 13 consists of primers shown by SEQ ID NO.29-30, group 14 consists of primers shown by SEQ ID NO.31-33, group 15 consists of primers shown by SEQ ID NO.34-37, and group 16 consists of primers shown by SEQ ID NO. 38-39.
3. The primer combination according to claim 1 or 2, wherein a working solution is separately prepared for each primer combination, wherein the concentration of the primer of group 1 is 2.5 pmol/. mu.L, the concentration of the primer of group 2 is 1 pmol/. mu.L, the concentration of the primer of group 3 is 2 pmol/. mu.L, the concentration of the primer of group 4 is 2 pmol/. mu.L, the concentration of the primer of group 5 is 2.5 pmol/. mu.L, the concentration of the primer of group 6 is 1 pmol/. mu.L, the concentration of the primer of group 7 is 4 pmol/. mu.L, the concentration of the primer of group 8 is 3 pmol/. mu.L, the concentration of the primer of group 9 is 1 pmol/. mu.L, the concentration of the primer of group 10 is 2 pmol/. mu.L, the concentration of the primer of group 11 is 3 pmol/. mu.L, the concentration of the primer of group 12 is 2.5 pmol/. mu.L, the concentration of the primer of group 13 pmol/. mu, the concentration of the primer set 14 was 1 pmol/. mu.L, the concentration of the primer set 15 was 1 pmol/. mu.L, and the concentration of the primer set 16 was 0.4 pmol/. mu.L.
4. A kit for KIR genotyping, comprising the primer combination of any one of claims 1-3.
5. Use of the primer combination of any one of claims 1-3 in the preparation of a kit for KIR genotyping.
6. Use of the primer combination of any one of claims 1-3, or the kit of claim 4, for KIR genotyping.
7. A method for KIR genotyping, comprising the steps of:
(1) obtaining DNA of a sample to be detected;
(2) performing PCR amplification using the primer combination according to any one of claims 1 to 3 or the kit according to claim 4, using the DNA obtained in step (1) as a template;
(3) and judging the KIR genotype of the sample to be detected according to the amplification result.
8. The method according to claim 7, wherein a working solution is separately prepared for each set of primer combination, wherein the concentration of the primer of group 1 is 2.5pmol/μ L, the concentration of the primer of group 2 is 1pmol/μ L, the concentration of the primer of group 3 is 2pmol/μ L, the concentration of the primer of group 4 is 2pmol/μ L, the concentration of the primer of group 5 is 2.5pmol/μ L, the concentration of the primer of group 6 is 1pmol/μ L, the concentration of the primer of group 7 is 4pmol/μ L, the concentration of the primer of group 8 is 3pmol/μ L, the concentration of the primer of group 9 is 1pmol/μ L, the concentration of the primer of group 10 is 2pmol/μ L, the concentration of the primer of group 11 is 3pmol/μ L, the concentration of the primer of group 12 is 2.5pmol/μ L, the concentration of the primer of group 13 is 12pmol/μ L, and the concentration of the primer of group 14 is 1pmol/μ L, the concentration of the primer in group 15 was 1 pmol/. mu.L, and the concentration of the primer in group 16 was 0.4 pmol/. mu.L.
9. The method of claim 7 or 8, wherein the PCR amplification system is:
mu.L PCR buffer, 1. mu.L 2.5mM dNTPs, 0.4. mu.L control, 1. mu.L primer combination, 1. mu.L cresol red, 1. mu.L sample DNA at 10-30 ng/. mu.L, 0.07. mu.L Taq enzyme with DNase-Free ddH2O made up to 10. mu.L of the reaction system.
10. The method of any one of claims 7-9, wherein the PCR amplification conditions are:
96 ℃ for 2 minutes, followed by 10 cycles of 94 ℃ for 10 seconds and 65 ℃ for 60 seconds, followed by 20 cycles of 94 ℃ for 10 seconds, 61 ℃ for 50 seconds, 72 ℃ for 30 seconds, and 15 ℃ in this order.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807387A (en) * | 2022-05-11 | 2022-07-29 | 深圳市血液中心(深圳市输血医学研究所) | KIR genotyping detection primer group and kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575292A (en) * | 2009-09-22 | 2012-07-11 | 霍夫曼-拉罗奇有限公司 | Determination of kir haplotypes associated with disease |
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| CN114807387A (en) * | 2022-05-11 | 2022-07-29 | 深圳市血液中心(深圳市输血医学研究所) | KIR genotyping detection primer group and kit |
| CN114807387B (en) * | 2022-05-11 | 2023-03-10 | 深圳市血液中心(深圳市输血医学研究所) | KIR genotyping detection primer group and kit |
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