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Method for genotyping HLA (Human Leucocyte Antigen) by using serum trace genome DNA

A genotyping and serum technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of low genomic DNA content, difficult to obtain and store samples, high sensitivity and specificity requirements, and achieve high sensitivity. High efficiency, simple and time-saving identification, and low sample consumption

Active Publication Date: 2010-12-15
北京科卫临床诊断试剂有限公司
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AI Technical Summary

Problems solved by technology

[0004] However, these three genotyping methods all require the fresh anticoagulated whole blood of the subject as the test sample, or extract it from the peripheral blood mononuclear cells (PBMC) isolated from the subject, and the samples are difficult to obtain and store
In contrast, serum is a relatively easy-to-obtain specimen, but the content of genomic DNA in serum is very low. As a sample for testing and typing, it is very technically difficult, and the requirements for sensitivity and specificity are quite high.

Method used

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  • Method for genotyping HLA (Human Leucocyte Antigen) by using serum trace genome DNA
  • Method for genotyping HLA (Human Leucocyte Antigen) by using serum trace genome DNA
  • Method for genotyping HLA (Human Leucocyte Antigen) by using serum trace genome DNA

Examples

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Embodiment 1

[0024] Example 1: Nested PCR technology amplifies HLA-A2, A11 and A24 genes in serum for genotyping

[0025] Amplification primer design: Select 114 sequences containing exon 2 and exon 3 of HLA-A gene from GenBank, mainly including Chinese-specific HLA-A11, HLA-A2 and HLA-A24 gene sequences, use Vector NTI software compares and analyzes HLA-A gene sequences in intron 1 and intron 3, and designs the first round of PCR general primers as follows: the length of the first round product fragment is 646bp, including exon 2 and exon 3 In the second round, specific upstream and downstream primers were designed according to the different SNPs between different genotypes in the exons, and the primers amplified PCR fragments of different lengths to distinguish the genotypes of HLA-A. Due to the differences in A2 and A24 PCR products Above 100bp can be well separated on agarose gel electrophoresis, so we put A2 and A24 in the same PCR tube, which saves cost and time better and greatly im...

Embodiment 2

[0074] Example 2 Nested PCR Detection of HLA-A Genotyping in Serum of Patients with HBV Infection

[0075] From 2005 to 2007, the serum samples of 709 patients with hepatitis B who visited the 302nd Hospital of the People's Liberation Army were analyzed for HLA-A genotype by serum trace PCR. The analysis results are as follows:

[0076] Case: The diagnostic criteria of hepatitis B conformed to the 2000 Xi’an viral hepatitis diagnostic criteria [Chinese Society of Infectious Diseases and Parasitic Diseases; Chinese Liver Society: Viral Hepatitis Diagnostic and Treatment Standards. Chinese Journal of Hepatology 2000; 6: 324 -329.], among the 709 patients, there were 294 cases of acute hepatitis B (AHB), 286 cases of chronic hepatitis (CHB), and 129 cases of chronic severe hepatitis (ACLF); male and female were 240 / 54, 252 / 34 and 106 / 23 cases, the average ages were 36.35±12.39, 39.92±12.69 and 45.88±12.10 years old. The positive rates of HBeAg were 25.7%, 59.0% and 35.4%, respec...

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Abstract

The invention discloses a method for genotyping an HLA (Human Leucocyte Antigen) by using a serum trace genome DNA with serum as a sample, comprising the following steps of: cracking and extracting DNA from small amount of serum and designing a specific primer; subjecting to two-turn net-PCR applying a net-PCR (Polymerase Chain Reaction) technology; and identifying three genotypes of HLA-A through electrophoresis by successfully using the sizes of PCR product segments. By using the serum as the analysis sample, the invention greatly increases convenience to genotyping the HLA.

Description

Technical field: [0001] The invention relates to a method of using serum as a sample to carry out HLA genotyping using serum trace genomic DNA, in particular to the application of sequence-specific primer polymerase chain reaction (SSP-PCR) to perform human HLA genotyping from serum trace genomic DNA. Method for Genotyping HLA-A2, A11 and A24 Leukocyte Antigen Class I Molecules (HLA-I). Background technique: [0002] Human leukocyte antigen (human leukocyte antigen, HLA) gene is a group of closely linked genes located on the short arm of human chromosome 6. These gene coding products not only determine the host's histocompatibility, but also participate in the host's immune response and immune regulation. For example, the type of infection that directly affects patients with hepatitis B virus (HBV) infection. [0003] The traditional HLA antigen typing method is a small amount of lymphocytotoxicity test, which has a high error rate and is expensive. It requires expensive f...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 徐东平刘妍许智慧李晓东钟彦伟戴久增王琳王耀
Owner 北京科卫临床诊断试剂有限公司
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