CN117925802B

CN117925802B - Primer composition for HLA-I and HPA multiplex PCR, application and genotyping method

Info

Publication number: CN117925802B
Application number: CN202410285101.1A
Authority: CN
Inventors: 和艳敏; 朱发明; 王芳; 洪小珍
Original assignee: BLOOD CENTER OF ZHEJIANG PROVINCE
Current assignee: BLOOD CENTER OF ZHEJIANG PROVINCE
Priority date: 2024-03-13
Filing date: 2024-03-13
Publication date: 2024-07-26
Anticipated expiration: 2044-03-13
Also published as: CN117925802A

Abstract

The invention provides a primer composition for HLA-I and HPA multiplex PCR, application and a genotyping method, and relates to the technical field of biology. The primer composition for HLA-I and HPA multiplex PCR comprises primer combinations with nucleotide sequences shown as SEQ ID NO. 1-20 respectively, multiplex PCR is carried out based on the primer, library construction sequencing is carried out on amplified products, bioinformatics analysis is carried out on the amplified products, genotypes of the HLA-I and the HPA in a sample to be detected are obtained, and the primer composition has the advantages of high throughput, rapidness and accuracy, can effectively reduce the genotyping cost of the HLA-I and the HPA, and saves the detection experiment time.

Description

Primer composition for HLA-I and HPA multiplex PCR, application and genotyping method

Technical Field

The invention relates to the field of biotechnology, in particular to a primer composition for HLA-I and HPA multiplex PCR, application and a genotyping method.

Background

Platelets have multiple functions of adhesion, release, aggregation and the like, and play an important role in the hemostatic and blood coagulation processes. Platelet infusion can be used as a support means for thrombocytopenia, hemorrhagic diseases, radiotherapy and chemotherapy of malignant tumor, leukemia and hematopoietic stem cell transplantation. Platelet antibodies are easily produced after multiple infusions of platelets, resulting in ineffective infusions. Factors that lead to ineffective platelet infusion include immune factors and non-immune factors. Non-immune factors include fever, infection, DIC (DISSEMINATED INTRAVASCULAR COAGULATION ), splenomegaly, and the like.

Immune PTR (platelet transfusion refractoriness, ineffective platelet infusion) is mainly associated with HLA-class I antibodies and HPA antibodies. Platelet surface presents a large number of antigens and are generally classified into two categories: one class is antigens shared with other blood cells or tissues, called platelet-associated antigens, such as HLA-I class antigens, ABO antigen systems, etc.; the other is a platelet-specific antigen, which shows a unique genetic polymorphism of platelets, namely a Human platelet antigen (Human PLATELET ANTIGEN, HPA), is an epitope located on the platelet membrane glycoprotein (glycoprotein, GP). However, the current platelet infusion only considers ABO homotype infusion, so that repeated platelet infusion can generate alloimmune antibodies, and further the platelet infusion is ineffective.

The most common method for solving the immune PTR at present is to perform random platelet serology cross matching, and select platelets matched with the serology matching for infusion; another method is platelet genotyping, i.e. selecting donor platelets matched with the patient's HLA-I and/or HPA genotype for infusion, which requires the establishment of a platelet donor gene database of known HLA-I, HPA genotyping data, selecting donor platelets matched with HLA-I, HPA genes for infusion, solving the problem of ineffective immune platelet infusion.

To build a platelet donor pool of known HLA and HPA genotyping data requires the establishment of accurate, convenient, high throughput HLA and HPA genotyping techniques. The current methods for detecting HLA and HPA mainly comprise PCR-SSP, PCR-SSO, PCR-SBT, new Generation Sequencing (NGS) and the like, and the methods of PCR-SSP and PCR-SSO in the methods are simple and quick, but have the problem of low accuracy; although the PCR-SBT method has high accuracy, the workload of the detection process is larger, the time is longer, and in addition, the methods can only detect HLA-I and HPA genes independently and cannot realize synchronous detection; while the latest NGS technology based on the capturing method can realize synchronous high-throughput detection of HLA and HPA, the existing method is not beneficial to quickly constructing a platelet donor gene database because of the high detection cost due to the design and synthesis of a plurality of probes, and a new detection strategy and method need to be found.

In view of this, the present invention has been made.

Disclosure of Invention

The present invention aims to provide a primer composition for HLA-I and HPA multiplex PCR to improve HLA and HPA genotyping methods, based on which the present invention also aims to provide its use and a method of HLA-I and HPA genotyping.

In order to solve the technical problems, the invention adopts the following technical scheme:

In a first aspect, there is provided a primer composition for HLA-I and HPA multiplex PCR comprising:

HLA-A primer pair: comprises a primer HLA-AF with a nucleotide sequence shown as SEQ ID NO.1 and a primer HLA-AR with a nucleotide sequence shown as SEQ ID NO. 2;

an HLA-B primer pair comprises a primer HLA-BF with a nucleotide sequence shown as SEQ ID NO.3 and a primer HLA-BR with a nucleotide sequence shown as SEQ ID NO. 4;

an HLA-C primer pair comprises a primer HLA-CF with a nucleotide sequence shown as SEQ ID NO.5 and a primer HLA-CR with a nucleotide sequence shown as SEQ ID NO. 6;

An HPA-1,4 primer pair comprising a primer HPA-1,4F with a nucleotide sequence shown as SEQ ID NO.7 and a primer HPA-1,4R with a nucleotide sequence shown as SEQ ID NO. 8;

An HPA-2 primer pair comprises a primer HPA-2F with a nucleotide sequence shown as SEQ ID NO.9 and a primer HPA-2R with a nucleotide sequence shown as SEQ ID NO. 10;

An HPA-3 primer pair comprises a primer HPA-3F with a nucleotide sequence shown as SEQ ID NO.11 and a primer HPA-3R with a nucleotide sequence shown as SEQ ID NO. 12;

an HPA-5 primer pair comprises a primer HPA-5F with a nucleotide sequence shown as SEQ ID NO.13 and a primer HPA-5R with a nucleotide sequence shown as SEQ ID NO. 14;

an HPA-6w primer pair comprising a primer HPA-6wF with a nucleotide sequence shown as SEQ ID NO.15 and a primer HPA-6wR with a nucleotide sequence shown as SEQ ID NO. 16;

an HPA-15 primer pair comprises a primer HPA-15F with a nucleotide sequence shown as SEQ ID NO.17 and a primer HPA-15R with a nucleotide sequence shown as SEQ ID NO. 18;

An HPA-21w primer pair comprising a primer HPA-21wF having a nucleotide sequence shown as SEQ ID NO.19 and a primer HPA-21wR having a nucleotide sequence shown as SEQ ID NO. 20.

In a second aspect, there is provided the use of the primer composition of the first aspect in any one of the following (I) to (V):

Amplification of HLA-I and HPA genes or preparation of a kit for amplification of HLA-I and HPA genes;

(II) constructing HLA-I and HPA gene sequencing libraries or preparing a kit for constructing the HLA-I and HPA gene sequencing libraries;

(III) used for HLA-I and HPA gene sequencing or preparing HLA-I and HPA gene sequencing kit;

(IV) used for HLA-I and HPA genotyping or preparing HLA-I and HPA genotyping kit;

(V) preparation of a platelet genotyping kit.

In a third aspect, there is provided a kit for HLA-I and HPA amplification, the kit comprising the primer composition of the first aspect.

In a fourth aspect, there is provided a method of HLA-I and HPA genotyping comprising amplifying HLA-I and HPA genes in a sample to be tested using the primer composition of the first aspect; constructing an amplification product as a sequencing library, sequencing and performing bioinformatics analysis on sequencing data to obtain genotypes of HLA-I and HPA in a sample to be detected;

the amplification is multiplex PCR amplification;

HLA-class I antigens include HLA-A, HLA-B and HLA-C; HPA antigens include HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w.

Compared with the prior art, the invention has the following beneficial effects:

According to the invention, by designing the specific primers, HLA-I and HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes are amplified simultaneously in the same system, so that the detection cost can be effectively reduced, the detection experiment time can be saved, and the emergency requirement of patients can be met. And it was verified that the HLA-I and HPA typing was 100% in conformity with the known typed samples after sequencing of the amplification products using the primer composition provided by the present invention.

The HLA-I and HPA genotyping method provided by the invention combines a sequencing technology on the basis of a multiplex PCR method, and has the characteristics of high throughput, rapidness and accuracy, and synchronous detection of HLA-I and HPA genes. The method is favorable for improving the detection efficiency of the HLA and HPA genes of the platelet donor gene database, and can be applied to the construction of the platelet donor gene database so as to expand the scale of the platelet donor gene database and the cooperation of genes, improve the safety and the effectiveness of platelet infusion, save platelet resources and improve the blood safety.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the electrophoresis results of a high throughput nucleic acid protein analysis system for multiplex PCR products Qsep400,400 of example 1;

FIG. 2 shows the results of library quality detection using Alignet 4200 electrophoresis apparatus of example 1;

FIG. 3 is a sample HLA-I typing result by TSV software in example 1;

FIG. 4a is the result of HPA-1 typing of a sample by CLC software in example 1;

FIG. 4b is the result of HPA-2 typing of a sample by CLC software in example 1;

FIG. 4c is the HPA-3 typing result of the sample by CLC software in example 1;

FIG. 4d is the HPA-4 typing result of the sample by CLC software in example 1;

FIG. 4e is the HPA-5 typing result of the sample by CLC software in example 1;

FIG. 4f is the result of HPA-6w typing of a sample by CLC software in example 1;

FIG. 4g HPA-15 typing results of samples by CLC software in example 1;

FIG. 4h HPA-21w typing of samples by CLC software in example 1;

FIG. 5 shows the NGS sequencing results at final concentrations of 25nM for HPA-1,4F and HPA-1,4R primers in example 2;

FIG. 6 shows the NGS sequencing results at final concentrations of 30nM for HPA-1,4F and HPA-1,4R primers in example 2;

FIG. 7 shows the NGS sequencing results at final HPA-5F and HPA-5R primer concentrations of 25nM in example 3;

FIG. 8 shows the NGS sequencing results at a final concentration of 30nM for HPA-5F and HPA-5R primers in example 3;

FIG. 9 shows the result of QIAxcel electrophoresis of the multiplex PCR product of comparative example 1;

FIG. 10 is a graph showing the typing results of HLA class I molecules from the NGS sequencing results of comparative example 1.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are 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.

In a first aspect, there is provided a primer composition for HLA-I and HPA multiplex PCR comprising the following primer pairs:

The HLA-A primer pair, the target gene is HLA-A gene, and is used for HLA-A typing, and the primer pair comprises a primer HLA-AF with a nucleotide sequence shown as SEQ ID NO.1 and a primer HLA-AR with a nucleotide sequence shown as SEQ ID NO. 2;

An HLA-B primer pair, wherein the target gene is an HLA-B gene and is used for HLA-B typing, and the HLA-B primer pair comprises a primer HLA-BF with a nucleotide sequence shown as SEQ ID NO.3 and a primer HLA-BR with a nucleotide sequence shown as SEQ ID NO. 4;

An HLA-C primer pair, wherein the target gene is an HLA-C gene and is used for HLA-C typing, and the HLA-C primer pair comprises a primer HLA-CF with a nucleotide sequence shown as SEQ ID NO.5 and a primer HLA-CR with a nucleotide sequence shown as SEQ ID NO. 6;

The HPA-1,4 primer pair, the target gene is ITGB3 gene, is used for HPA-1 and HPA-4 typing, and comprises a primer HPA-1,4F with a nucleotide sequence shown as SEQ ID NO.7 and a primer HPA-1,4R with a nucleotide sequence shown as SEQ ID NO. 8;

The HPA-2 primer pair, the target gene is GP1BA gene, is used for HPA-2 typing, and comprises a primer HPA-2F with a nucleotide sequence shown as SEQ ID NO.9 and a primer HPA-2R with a nucleotide sequence shown as SEQ ID NO. 10;

An HPA-3 primer pair, wherein the target gene is ITGA2B gene, which is used for HPA-3 typing and comprises a primer HPA-3F with a nucleotide sequence shown as SEQ ID NO.11 and a primer HPA-3R with a nucleotide sequence shown as SEQ ID NO. 12;

An HPA-5 primer pair, wherein the target gene is ITGA2 gene, which is used for HPA-5 typing and comprises a primer HPA-5F with a nucleotide sequence shown as SEQ ID NO.13 and a primer HPA-5R with a nucleotide sequence shown as SEQ ID NO. 14;

The HPA-6w primer pair, the target gene is ITGB3 gene, is used for HPA-6w typing, and comprises a primer HPA-6wF with a nucleotide sequence shown as SEQ ID NO.15 and a primer HPA-6wR with a nucleotide sequence shown as SEQ ID NO. 16;

The HPA-15 primer pair, the target gene is CD109 gene, is used for HPA-15 typing, and comprises a primer HPA-15F with a nucleotide sequence shown as SEQ ID NO.17 and a primer HPA-15R with a nucleotide sequence shown as SEQ ID NO. 18;

the primer pair of HPA-21w, the target gene is ITGB3 gene, is used for HPA-21w typing, and comprises a primer HPA-21wF with a nucleotide sequence shown as SEQ ID NO.19 and a primer HPA-21wR with a nucleotide sequence shown as SEQ ID NO. 20.

In an alternative embodiment, the working concentration of each primer is:

HLA-AF is 100 to 400nm, which may be, for example but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HLA-AR is 100 to 400nm, which may be, for example but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HLA-BF is 100 to 400nm, and may be, for example but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HLA-BR is 100 to 400nm, for example, but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HLA-CF is 100 to 400nm, which may be, for example but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HLA-CR is 100-400 nm, and may be, for example but not limited to, 100, 150, 200, 250, 300, 350 or 400nm, preferably 200nm; HPA-1,4F is 50 to 200nm, which may be, for example but not limited to, 50, 60, 80, 100, 120, 150 or 200nm, preferably 60nm; HPA-1,4R is 50 to 200nm, which may be, for example but not limited to, 50, 60, 80, 100, 120, 150 or 200nm, preferably 60nm; HPA-2F is 10 to 100nm, and may be, for example but not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 25nm; HPA-2R is 10 to 100nm, and may be, for example but not limited to, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 25nm; HPA-3F is 20 to 100nm, and may be, for example but not limited to, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 40nm; HPA-3R is 20 to 100nm, and may be, for example but not limited to, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 40nm; HPA-5F is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 50nm; HPA-5R is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 50nm; HPA-6wF is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 55nm; HPA-6wR is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 55nm; HPA-15F is 20 to 100nm, and may be, for example but not limited to, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 40nm; HPA-15R is 20 to 100nm, and may be, for example but not limited to, 20, 30, 40, 50, 60, 70, 80, 90 or 100nm, preferably 40nm; HPA-21wF is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 55nm; HPA-21wR is 40 to 100nm, and may be, for example but not limited to, 40, 50, 60, 70, 80, 90 or 100nm, preferably 55nm.

In a preferred embodiment, the working concentration of each primer is: HLA-AF, HLA-AR, HLA-BF, HLA-BR, HLA-CF and HLA-CR are 200nm; HPA-1,4F and HPA-1,4R are 60nm; HPA-2F and HPA-2R are 25nm; HPA-3F, HPA-3R, HPA-15F and HPA-15R are 40nm; HPA-5F and HPA-5R are 50nm; and HPA-6wF, HPA-6wR, HPA-21wF and HPA-21wR are 55nm.

In a second aspect, there is also provided the use of the primer composition of the first aspect in any one of the following (I) to (V):

and (I) amplifying HLA-I and HPA genes or preparing a kit for amplifying HLA-I and HPA genes.

(II) constructing HLA-I and HPA gene sequencing library or preparing a kit for constructing HLA-I and HPA gene sequencing library.

(III) used for HLA-I and HPA gene sequencing or preparing HLA-I and HPA gene sequencing kit.

(IV) for HLA-I and HPA genotyping or preparing an HLA-I and HPA genotyping kit, wherein HLA-I and HPA genotyping is non-diagnostic and therapeutic destination.

(V) preparation of a platelet genotyping kit.

In a third aspect, there is also provided a kit for HLA-I and HPA amplification, the kit comprising the primer composition of the first aspect.

The kit of the second and third aspects comprises the primer composition of the first aspect and optionally further comprises one or more of conventional kits known in the art, including but not limited to enzymes for amplification, buffers, dNTPs, reagents for DNA extraction, reagents for purification of DNA samples, reagents for library construction or sequencing and solid phase carriers, and the skilled person can select the composition of reagents in the product according to the actual detection means, which is not limiting in the present invention.

In a fourth aspect, there is also provided a method of HLA-I and HPA genotyping, the method comprising amplifying HLA-I and HPA genes in a sample to be tested using the primer composition of the first aspect; the amplification products are constructed as a sequencing library, and then sequencing and bioinformatic analysis are carried out on sequencing data to obtain genotypes of HLA-I and HPA in a sample to be tested, wherein the amplification is multiplex PCR amplification on the template by using the primer composition of the first aspect.

In the genotyping method, HLA-I includes HLA-A, HLA-B and HLA-C; HPA includes HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w.

In an alternative embodiment, the method uses genomic DNA as an amplification template.

In an alternative embodiment, the annealing temperature is 60℃in the multiplex PCR amplification reaction procedure.

In an alternative embodiment, the amplified sample is purified using a magnetic bead method.

In an alternative embodiment, the purified product is subjected to Fluoroskan DNA double-stranded quantitative analysis.

In an alternative embodiment, library preparation is performed using a transposon enzyme method.

In alternative embodiments, second generation sequencing is used to obtain sequencing data.

In alternative embodiments, HLA-A, HLA-B, and HLA-C typing is performed using software TYPESTREAM VISUAL SOFTWARE; HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w typing was performed using software CLC Main Workbench.

In a specific embodiment, the method of HLA-I and HPA genotyping comprises:

Specific primers for HLA-I class (HLA-A, HLA-B and HLA-C) and HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes were designed and synthesized.

(A) Human genomic DNA was prepared as a template for multiplex PCR amplification, and the amount of the DNA template was preferably 30 ng/. Mu.L.

(B) The primer compositions of the first aspect were used in the same tube to amplify fragments of interest of HLA-I class (HLA-A, HLA-B and HLA-C) and HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes by multiplex PCR under the same conditions by adjusting the concentrations of the different primers.

The multiplex PCR system is preferably formulated as follows: 4.0. Mu.L of 5 XGLX PCR buffer; 2.5mM dNTP 1.6. Mu.L; HLA-AF, HLA-AR, HLA-BF, HLA-BR, HLA-CF and HLA-CR initial concentrations were adjusted to 10. Mu.M, HPA-1,4F and HPA-1,4R initial concentrations were adjusted to 3.0. Mu.M, HPA-2F and HPA-2R initial concentrations were adjusted to 1.25. Mu.M, HPA-3F and HPA-3R initial concentrations were adjusted to 2.0. Mu.M, HPA-5F and HPA-5R initial concentrations were adjusted to 2.5. Mu.M, HPA-6wF and HPA-6wR initial concentrations were adjusted to 2.75. Mu.M, HPA-15F and HPA-15R initial concentrations were adjusted to 2.0. Mu.M, HPA-21wF and HPA-21wR initial concentrations were adjusted to 2.75. Mu.M, and 0.4. Mu.L for each primer; 1.25U/. Mu.L GLX-Taq enzyme 0.8. Mu.L; 4.0. Mu.L of DNA; the H2O 1.6. Mu.L was used to make up to 20. Mu.L.

The PCR reaction procedure is preferably: pre-denaturation at 94℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 68℃for 3min,30 cycles, extension at 68℃for 10min, and cooling to 12 ℃.

(C) Purifying the amplified product obtained in step (B) by a magnetic bead method, wherein the magnetic beads are preferablyThe XP beads and purification process is preferably automated on a Hamilton automated loading instrument.

(D) And (C) performing double-strand quantitative analysis of the Fluoskan DNA, wherein the double-strand quantitative reagent of Fluoskan DNA is preferably Quant-iT PicoGreen DSDNA ASSAY KIT.

(E) Library preparation is carried out on the purified product quantified in the step (D) by adopting a transposon enzyme method, and the method comprises the steps of fragmentation of amplified products, PCR enrichment and length sorting of the amplified products. The library preparation reagent is preferably TruePrep Flexible DNA Library Prep kit for Illumina and the initial amount of product after purification is preferably 100ng.

(F) The library obtained in step (E) was subjected to mass detection by an align 4200 electrophoresis apparatus, the fragment size of the library was analyzed, and library mixing was performed after quantitative analysis by Fluoroskan DNA double strand. The library quality detection reagent is preferably HIGH SENSITIVITY D1000 electrophoresis reagents.

(G) And (3) carrying out Illumina Miseq-platform sequencing on the mixed library obtained in the step (F). The Sequencing reagent is preferably Miseq Sequencing REAGENT KIT (v 2,300 cycles).

(H) And (3) typing the FASTQ original data obtained in the step (G) by using HLA typing special data software TYPESTREAM VISUAL SOFTWARE VERSION 2.0.0 for HLA-I type (HLA-A, HLA-B and HLA-C), performing bioinformatics analysis by using CLC Main Workbench 23.0.0 software special software, and designating individual HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genotypes.

The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.

Example 1

The parting method comprises the following steps:

1. Specific primers 20 were designed and synthesized for HLA-I class (HLA-A, HLA-B and HLA-C) and HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes, the sequences of the primers are shown in Table 1 below, and the amplified primers were diluted with pure water to a stock concentration of 50. Mu.M.

TABLE 1 primer information

2. 188 Human genomic DNA was prepared as a template for PCR amplification in the subsequent step.

188 Samples of known HLA-I class and HPA system typing in Zhejiang save blood platelet gene database (analytical method see Table 5) were selected, 400. Mu.L of whole blood to be tested was taken, genomic DNA was extracted according to MagDNA Pure LC DNA Isolation Kit kit instructions, and the concentration and purity of genomic DNA were determined.

3. Multiplex PCR simultaneously amplifies specific fragments of HLA-I class (HLA-A, HLA-B and HLA-C) and HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes.

Preparing GXL-Taq enzyme (TaKaRa), 5 XGXL buffer, dNTP (TaKaRa), pure water and the genome DNA template prepared in the step 2, diluting 20 primers in the step 1 to working concentration according to the concentration of the table 2, and preparing a PCR amplification system according to the system shown in the table 3 for each sample.

TABLE 2 working concentrations of different primers

TABLE 3 multiplex PCR reaction System

The PCR instrument (ABI 9700) was amplified according to the following procedure:

Pre-denaturation at 94℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 15s, extension at 68℃for 3min,30 cycles, extension at 68℃for 10min, and cooling to 12 ℃.

4. And (5) purifying the amplified product by a magnetic bead method.

By usingXP magnetic beads were used to purify multiplex PCR amplification products, performed on a Hamilton automated sample applicator. 94 samples were purified on a 96-well amplification plate at a time, 2 samples were blank, and 188 samples were performed in duplicate. The brief description is as follows: mu.l of each well of a 96-well PCR amplification plate was addedXP reagent, shake mix after room temperature place 5 minutes, put 96 hole PCR amplification board on the magnetic force frame until solution becomes clear, discard supernatant, add fresh 80% alcohol 150 μl to wash twice in each hole, after alcohol volatilize completely, add 27 μl TE solution to carry on the magnetic bead to dissolve in each hole, place 5 minutes after room temperature place on the magnetic force frame until solution becomes clear, finally absorb 25 μl supernatant to the new 96 hole PCR amplification board in each hole.

5. And (5) accurately quantifying the purified product.

The purified multiplex PCR amplified products were quantified using Quant-iT PicoGreen DSDNA ASSAY KIT, respectively.

6. Library preparation was performed using the transposon enzyme method.

Library preparation was performed on purified product starting at 100ng using TruePrep Flexible DNA Library Prep kit for Illumina reagents, and was performed strictly according to the instructions, including DNA fragmentation, PCR enrichment, and amplified product length sorting.

7. The obtained library was subjected to quality detection by an align 4200 electrophoresis apparatus.

And (3) carrying out electrophoresis on the library obtained in the step (6) by adopting HIGH SENSITIVITY D1000,1000 electrophoresis reagents and an align 4200 electrophoresis apparatus, and analyzing the size of the sorted fragments, wherein the main peak of the length of the sorted fragments is 500-550 bp.

8. And (5) quantitatively feeding the mixed library to a Illumina Miseq-platform sequencing machine.

After the double-stranded quantification of the Fluoskan DNA of the library obtained in the step 6 is carried out, the lowest concentration value in 188 samples is taken as a target dilution value, the total amount of each sample is taken to be 14 mu L, the amount of each sample and the amount of added Low TE solution are calculated, 188 samples are mixed into a tube, the molar concentration of the final library is calculated, the concentration of the library is adjusted to be 4nM, the double-stranded library is denatured into a single-stranded library through 0.2N NaOH, finally the upper machine concentration of the library is adjusted to be 20pM by utilizing HT1 precooled at 4 ℃ and the upper machine sequencing is carried out on sample holes of a MISEQ REAGENT KIT V kit.

9. Bioinformatics analysis.

Performing HLA-I (HLA-A, HLA-B and HLA-C) typing on the derived FASTQ original data by adopting HLA typing special data software TYPESTREAM VISUAL SOFTWARE VERSION 2.0.0 (TSV 2.0); analyzing by CLC Main Workbench 23.0.0 software (CLC) special software, removing sequences with low quality and repeated sequences according to quality evaluation of sequencing data, splicing all effective read lengths (reads) according to HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w gene standard sequences, rearranging the spliced sequences, and finally analyzing polymorphism of the determined sequences, and designating individuals according to HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w gene polymorphism conditions and reference HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w allele standard sequences.

(II) experimental results:

1. the multiplex PCR products were electrophoresed using Qsep.sup.400 high throughput nucleic acid protein analysis system and the results are shown in FIG. 1, which shows peak shapes of 1706bp, 1965bp, 2122bp, 2558bp, 3065 bp. The result of the quality detection of the Alignet 4200 electrophoresis apparatus library is shown in FIG. 2, and the main peak of the fragment after fragment sorting is about 500 bp.

The typing result coincidence rate of 2.188 samples is 100%. Taking polymorphism of one sample as an example, carrying out HLA-I typing on the sample by TSV2.0 software, wherein the result is shown in figure 3, and the sample result is HLA-A 02:01, -A26:01; HLA-B13:01, -B35:02; HLA-C.times.03:04, -C.times.04:01. The HPA is typed by CLC, the polymorphic site of the allelic gene of the HPA system is shown in Table 4, the analysis result of the CLC is shown in fig. 4a to 4h, the variation site of the sample HPA-1 is c.176T/T, the variation site of the sample HPA-2 is c.1008C/T, the variation site of the sample HPA-3 is c.262T/G, the variation site of the sample HPA-4 is c.506G/G, the variation site of the sample HPA-5 is c.160G/G, the variation site of the sample HPA-6w is c.1544G/G, the variation site of the sample HPA-15 is c.2104G/A, the variation site of the sample HPA-21w is c.19621G/G, the sample HPA-21 aa-4 aa-1 ab-4, the result of the sample HPA-20 ab-4 aa-1 is about the HPA-20G, and the result of the sample aa-20 ab-4 h is about.

Table 4 HPA System allelic polymorphism loci

3. Further, 376 samples of known HLA-I typing and HPA typing of Zhejiang save blood platelet gene database are detected by the method, and the typing result coincidence rate is found to be 100%. The original method is compared with the method of the invention in detection time, detection cost and the like. As shown in Table 5

TABLE 5 comparison of example 1 method with the prior art method

Example 2

The difference from example 1 is only that the final concentrations of HPA-1,4F and HPA-1,4R primers are 25nM or 30nM, and the amplification results are shown in FIGS. 5 and 6, and it can be seen that reads are not detected when the final concentrations of HPA-1,4F and HPA-1,4R primers are 25 nM; at a final concentration of 30nM, the ratio of ITGB3 gene reads was very low.

Example 3

The difference from example 1 was only that the final concentrations of HPA-5F and HPA-5R primers were 25nM or 30nM, and the amplification results are shown in FIGS. 7 and 8, and it can be seen that only 1 reads were detected when the final concentrations of HPA-5F and HPA-5R primers were 25 nM; at a final concentration of 30nM, the coverage of ITGA2 gene reads was low.

Comparative example 1

Primers for amplifying HLA-I class (HLA-A, HLA-B and HLA-C) genes were the same as in example 1, and primers for amplifying HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w genes were shown in Table 6, and multiplex PCR was performed on the samples, and the result of the electrophoretic amplification was shown in FIG. 9, whereby accurate amplified bands were obtained. A sequencing library was constructed, sequenced, and bioinformatic analyzed as described in example 1, and the NGS sequencing results showed that HLA-I class was not accurately typed (where HLA-A and HLA-C detected partial reads and HLA-B did not detect matched reads), as shown in FIG. 10.

Table 6 HPA comparative primer sequence listing

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A primer composition for HLA-I and HPA multiplex PCR, characterized in that the primer composition consists of the following primer pairs:

2. The primer composition of claim 1, wherein each primer has a working concentration of:

HLA-AF, HLA-AR, HLA-BF, HLA-BR, HLA-CF and HLA-CR are each independently 100 to 400nm;

HPA-1,4F and HPA-1,4R are respectively and independently 50-200 nm;

HPA-2F and HPA-2R are respectively and independently 10-100 nm;

HPA-3F, HPA-3R, HPA-15F and HPA-15R are respectively and independently 20-100 nm;

And HPA-5F, HPA-5R, HPA-6wF, HPA-6wR, HPA-21wF and HPA-21wR are each independently 40 to 100nm.

3. The primer composition of claim 2, wherein each primer has a working concentration of:

HLA-AF, HLA-AR, HLA-BF, HLA-BR, HLA-CF and HLA-CR are 200nm;

HPA-1,4F and HPA-1,4R are 60nm;

HPA-2F and HPA-2R are 25nm;

HPA-3F, HPA-3R, HPA-15F and HPA-15R are 40nm;

HPA-5F and HPA-5R are 50nm;

And HPA-6wF, HPA-6wR, HPA-21wF and HPA-21wR are 55nm.

4. Use of a primer composition according to any one of claims 1 to 3 for non-diagnostic and therapeutic purposes as defined in any one of (i) to (v):

(V) preparation of a platelet genotyping kit.

5. Kit for HLA-I and HPA amplification, characterized by comprising the primer composition according to any one of claims 1 to 3.

6. A method for genotyping HLA-I and HPA for non-diagnostic and therapeutic purposes, comprising amplifying HLA-I and HPA genes in a test sample using the primer composition of any one of claims 1 to 3; constructing an amplification product as a sequencing library, sequencing and performing bioinformatics analysis on sequencing data to obtain genotypes of HLA-I and HPA in a sample to be detected;

the amplification is multiplex PCR amplification;

7. The method of claim 6, wherein genomic DNA is used as an amplification template.

8. The method of claim 6, wherein the amplified sample is purified using a magnetic bead method and then a sequencing library is constructed.

9. The method according to claim 8, wherein the purified product is subjected to Fluoroskan DNA double-strand quantitative analysis.

10. The method of claim 6, wherein the library preparation is performed by a transposon enzyme method.

11. The method of claim 6, wherein sequencing data is obtained using second generation sequencing.

12. The method of claim 11, wherein the sequencing is performed using a Illumina Miseq platform.

13. The method of claim 6, wherein HLA-A, HLa-B, and HLa-C typing is performed using software TYPESTREAM VISUAL SOFTWARE; HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6w, HPA-15 and HPA-21w typing was performed using software CLC Main Workbench.