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Primer group, kit and method for HLA-DRB1 gene high-resolution typing

A HLA-DRB1, high-resolution technology, applied in the field of primer sets for HLA-DRB1 genotyping, can solve the problems of long full-length fragments, high cost, and high requirements for DNA template integrity

Active Publication Date: 2018-09-28
BEIJING NUOSHI KANGYING MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, the technical problem to be solved by the present invention is that the existing HLA-DRB1 gene full-length fragment is relatively long, the DNA template integrity requirement is high, the amplification is difficult, the cost is high, and it cannot meet the problem of large-scale sample HLA-DRB1 typing. The invention provides a primer set, kit and method for HLA-DRB1 gene amplification and high-resolution typing

Method used

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  • Primer group, kit and method for HLA-DRB1 gene high-resolution typing
  • Primer group, kit and method for HLA-DRB1 gene high-resolution typing
  • Primer group, kit and method for HLA-DRB1 gene high-resolution typing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1 Amplification of HLA-DRB1 gene

[0096] 1. Sample DNA Extraction

[0097] DNA was extracted from 96 blood samples with known HLA-DRB1 genotypes using the QlAamp blood extraction kit (QIAGEN). The concentration of the extracted DNA samples was measured by a UV spectrophotometer, and the concentration of the extracted DNA samples was adjusted to 20-50 ng / μl.

[0098] 2. Design HLA-DRB1 Gene Amplification Primers

[0099] According to the latest HLA-DRB1 gene sequence in the IMGT / HLA database (http: / / www.ebi.ac.uk / imgt / hla / ), such as figure 1 As shown, the HLA-DRB1 gene has 6 exons, and multiple groups (two in each group) of suitable conserved regions are searched, and it is ensured that each group of conserved regions can cover exons 2-3 of the HLA-DRB1 gene. Appropriate amplification primers are designed respectively in the found multiple groups of conserved regions. When several allele sequences are inconsistent, degenerate bases are used, or a new allele-s...

Embodiment 2

[0112] Example 2 Amplification of HLA-DRB1 gene

[0113] This example is basically the same as Example 1, except that the 5' end and / or 3' end of the first set of amplification primers are added with a sequence of less than or equal to 8 nucleotides, in this example, they are respectively in DRB1 - 5' and 3 of F2-1, DRB1-F2-2, DRB1-F2-3, DRB1-F2-4, DRB1-F2-5, DRB1-F2-6, DRB1-F2-7 and DRB1-2R 8 nucleotides are added at the ' end, and the nucleotides can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine). 5'→3' to increase GGCTACAG, 5'→3' to increase TTTGAACC, exon 2 of HLA-DRB1 gene can be amplified using the first set of amplification primers, and the 5' end of the second set of amplification primers And / or the 3' end is added with a sequence of less than or equal to 8 nucleotides, in this embodiment, it is a sequence with 1 nucleotide added at the 5' end and 3' end of DRB1-F3 and DRB1-R3, respectively, In this example, G is added at 5'→3' at the 5' end, ...

Embodiment 3

[0114] Example 3 Amplification of HLA-DRB1 gene

[0115] This example is basically the same as Example 1, except that the 5' end and / or 3' end of the first set of amplification primers are added with a sequence of less than or equal to 8 nucleotides, in this example, they are respectively in DRB1 - 5' and 3 of F2-1, DRB1-F2-2, DRB1-F2-3, DRB1-F2-4, DRB1-F2-5, DRB1-F2-6, DRB1-F2-7 and DRB1-2R 1 nucleotide is added to the ' end, the nucleotide can be selected from A (adenine), T (thymine), C (cytosine) or G (guanine), in this example, the 5' end is increased C, G is added at the 3' end, exon 2 of the HLA-DRB1 gene can be amplified using the first set of amplification primers, and the 5' end and / or 3' end of the second set of amplification primers are increased by less than or equal to 8 The sequence of nucleotides, in this example, is a sequence with 8 nucleotides added to the 5' and 3' ends of DRB1-F3 and DRB1-R3 respectively, in this example, the 5' → CACAGTGT was added to t...

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Abstract

The invention discloses a primer group, kit and method for HLA-DRB1 gene typing, and belongs to the field of gene detection. According to the primer group for HLA-DRB1 gene high-resolution typing, primers are designed correspondingly according to exons 2 and exons 3 of HLA-DRB1 genes, and the primers are utilized to conduct PCR amplification on the HLA-DRB1 genes. The primers conduct PCR amplification on the HLA-DRB1 genes, thus the gene fragment length of an amplified product is smaller than 1 kb, the requirement for the integrity of a template is reduced, the amplification efficiency is high, the amplification reaction time is short, the cost is reduced, the gene typing requirement of the large-scale sample HLA-DRB1 genes can be met, the more accurate basis is provided for clinical HLA matching, a proper transplant donor is provided for a patient, the rejection reaction in the transplant process is reduced, and the success rate of organ transplantation and the survival rate of the patient are increased.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a primer set, a kit and a method for HLA-DRB1 genotyping. Background technique [0002] Human leukocyte antigen (HLA), the encoding gene is located on the short arm of chromosome 6, with a total length of about 4000Kb. It is the most complex genetic polymorphism system known to humans, and is closely related to the function of the human immune system. HLA, also known as transplant antigen, is an important factor in determining the level of transplant rejection. During organ transplantation, the higher the degree of HLA compatibility between the donor and the recipient, the lower the incidence of rejection, the higher the success rate of transplantation and the long-term survival rate of transplanted organ patients; conversely, the more likely to reject reaction. Therefore, efficient and accurate typing of HLA is crucial for organ transplantation. [0003] HLA genes ar...

Claims

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Application Information

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IPC IPC(8): C12Q1/6881C12Q1/6869C12N15/11
Inventor 康颖
Owner BEIJING NUOSHI KANGYING MEDICAL TECH
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