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Human leukocyte antigen one-step sequencing typing method and application thereof

A technology for sequencing and sequencing primers, which can be used in biochemical equipment and methods, resistance to vector-borne diseases, and determination/examination of microorganisms.

Active Publication Date: 2021-04-06
BEIJING SEARCH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The main purpose of the present invention is to provide a simple and fast method for detecting HLA genotyping, and control the rech

Method used

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  • Human leukocyte antigen one-step sequencing typing method and application thereof
  • Human leukocyte antigen one-step sequencing typing method and application thereof
  • Human leukocyte antigen one-step sequencing typing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0039] Example 1

[0040] The PCR primers, sequencing primers and ambiguous typing primers provided in this example are shown in Table 1:

[0041] Table 1PCR primers, sequencing primers and ambiguous typing primers

[0042]

[0043]

[0044] Note: In the table, the letter K represents any nucleotide in T / G, the letter R represents any nucleotide in A / G, and the letter M represents any nucleotide in A / C. The letter S represents any nucleotide in C / G, and the letter Y represents any nucleotide in C / T; primers are designed to avoid sequence formation of high-level structures and dimers, and introduce correction bases to ensure Primers fit the purpose of design.

[0045] The primers in Table 1 were used to perform one-step sequencing and typing of human leukocyte antigen on the anticoagulated bone marrow or whole blood samples of 1,000 healthy individuals. The specific steps are as follows:

[0046] 1. Sample Preparation

[0047] 1.1. Sample processing and nucleic acid e...

Example Embodiment

[0122] Example 2

[0123] According to the primers and reagents used in Example 1, a kit was prepared. The kit includes specific HLA amplification primers, sequencing primers, high-frequency ambiguous discrimination primers, and PCR amplification and sequencing reagents. details as follows:

[0124] The main components of the kit

[0125] See Table 8 (see Table 1 for specific primer sequences):

[0126] Table 8 Main Components

[0127]

[0128]

[0129]

[0130]

[0131] Note: The components of this kit cannot be replaced between different batches, and cannot be replaced with corresponding components of other products.

[0132] In addition, this kit does not include but necessary equipment, reagents and consumables: PCR instrument, gene analyzer, nucleic acid extraction reagent, biological safety cabinet, high-speed refrigerated centrifuge, dry thermostat, vortex mixer, microcentrifuge, Pipette and tip, centrifuge tube, PCR reaction tube, 96-well PCR plate, ab...

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PUM

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Abstract

The invention provides an ambiguous typing primer group for HLA (human leukocyte antigen), a specific primer composition containing the ambiguous typing primer group and used for HLA sequencing typing, and a corresponding HLA genotyping method. According to the invention, the ratio of the common HLA sequencing typing method, which needs to be reviewed due to the type of the moire, is effectively controlled from 10-15% to below 0.1% by adding the ambiguous typing primer group, so that precious medical treatment time, labor cost and reagent cost are saved.

Description

technical field [0001] The invention specifically relates to a one-step sequencing and typing method for human leukocyte antigen and its application. Background technique [0002] The human leukocyte antigen (Human leukocyte antigen, HLA) coding gene is located on the short arm of chromosome 6, which is an important antigenic substance that constitutes transplant rejection. During bone marrow and organ transplantation, the HLA compatibility between donor and recipient is more The higher the value, the lower the incidence of rejection, the higher the success rate of transplantation and the long-term survival rate of transplanted organs; on the contrary, the rejection is more likely to occur. The key to the success of organ transplantation is to control graft rejection. At present, it is mainly predicted by the matching of HLA-A, HLA-B, HLA-C, HLA-DQB1, and HLA-DRB1 sites between the donor and the recipient before transplantation. Possible transplant rejection. With the deve...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6869C12N15/11
CPCC12Q1/6888C12Q1/6869C12Q2600/156C12Q2531/113Y02A50/30
Inventor 王志富郭同垒刘常峥赵敏方圆刘明坤叶锋
Owner BEIJING SEARCH BIOTECH
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