Human leucocyte antigen genotyping method and reagent thereof

A white blood cell antigen, genotyping technology, applied in HLA-B, HLA-C locus 5, 7 exon genotyping, 6 fields, can solve the problem that new alleles cannot be covered and identified, and positive bands Not clear enough, easy to cause misjudgment and other problems, to achieve the effect of easy identification and result interpretation, improved resolution and accuracy, and improved work efficiency

Inactive Publication Date: 2016-02-03
SHENZHEN BLOOD CENT
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

In the past, the PCR-SSP (polymerase chain reaction-sequence-specific primer) method used in the past, due to the rapid discovery of new alleles, the update of commercial PCR-SSP reagents lagged behind, and some new alleles Genes may not be covered and identified
Moreover, some positive bands are not clear enough or non-specific bands appear in some wells, which may easily cause misjudgment

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  • Human leucocyte antigen genotyping method and reagent thereof
  • Human leucocyte antigen genotyping method and reagent thereof
  • Human leucocyte antigen genotyping method and reagent thereof

Examples

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Embodiment 1

[0062] 150 samples with known HLA-B allele genotypes were randomly selected from clinical transplant matching samples. Use the PCR primers and sequencing primers of the present invention to carry out sequencing typing of ambiguous results to verify the accuracy of the typing method of the present invention.

[0063] First, the HLA-B gene was detected, and PCR amplification was performed strictly according to the instructions of the AlleleSEQRHLA Sequencing Kit (AtriaGenetics). The HLA-B amplification product has two product fragments, the lengths of which are 1.3kb and 1.5kb respectively. PCR amplification products were purified using ExoSAP-IT (shrimp alkaline phosphatase) to remove excess free PCR primers and substrate dNTPs; then the purified amplification products were used as templates for routine and unconventional detection areas HLA-B exons 5, 6, and 7 sequencing primers were used for sequencing reactions.

[0064] Sequencing reaction system of HLA-B unconventional d...

Embodiment 2

[0074] 150 samples with known HLA-C allele genotypes were randomly selected from clinical transplantation matching samples. Use the PCR primers and sequencing primers of the present invention to carry out sequencing typing of ambiguous results to verify the accuracy of the typing method of the present invention.

[0075] First, the HLA-C gene was detected, and PCR amplification was performed strictly according to the instructions of the AlleleSEQRHLA Sequencing Kit (AtriaGenetics). The HLA-C amplification product has two product fragments, the lengths of which are 1.3kb and 1.5kb respectively. PCR amplification products were purified using ExoSAP-IT (shrimp alkaline phosphatase) to remove excess free PCR primers and substrate dNTPs; then the purified amplification products were used as templates for routine and unconventional detection areas HLA-C exons 5, 6, and 7 sequencing primers were used for sequencing reactions.

[0076] Sequencing reaction system of HLA-C unconventio...

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Abstract

The invention discloses a human leucocyte antigen genotyping method and a reagent thereof. The method comprises the following steps: firstly, implementing PCR amplification to HLA-B and HLA-C site genes of to-be-detected genome DNA; and then, conducting a sequencing reaction to amplification products by virtue of 12 sequencing primers so as to determine a genotype, wherein the sequencing primers include 5th, 6th and 7th sequencing primers of an HLA-B exon and 5th, 6th and 7th sequencing primers of an HLA-C exon. The method disclosed by the invention, by virtue of additionally exon sequencing in an irregular detection area, can effectively solve the problem that many and many ambiguous results appear during HLA high-resolution genotyping. The obtained sequence is free from a background signal and a miscellaneous peak; and the sequence, which can be introduced into analysis software which is matched with the commercial reagent, is easy for recognition and result interpretation. The primers disclosed by the invention can undergo synchronous amplification and sequencing with a commercial kit, so as to improve the working efficiency and to significantly reduce the cost of the reagent; therefore, the invention is of very significant meaning in the improvement of the detection efficiency.

Description

technical field [0001] The invention relates to a genotyping method and reagent thereof, in particular to a method for genotyping of exons 5, 6 and 7 of HLA-B and HLA-C sites and the reagent thereof. Background technique [0002] The genetic region of the HLA complex is located on the short arm of human chromosome 6, with a total length of 4000kb, and its function is related to immune response. HLA molecules present antigenic peptides to T lymphocytes for recognition and play an extremely important role in adapting immune responses. The HLA gene is also an extremely important genetic structure of human beings, which is of great significance in the fields of organ transplant immune rejection and judicial identification. HLA genes are divided into class I, class II and class III. HLA-B, -C loci belong to class I genes. [0003] As of July 2014, there were 3455 and 2529 alleles of HLA-B and -C loci published in the International Immunogenetics IMGT / HLA Database. HLA gene se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q2600/112
Inventor 王大明
Owner SHENZHEN BLOOD CENT
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