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A real-time fluorescent PCR-based method for detecting the hla‑b*5801 allele

An allele and HLA-B technology, applied in the field of alleles, can solve problems such as difficult primer-probe combination, save experimental time and consumables, short probe sequences, and easy to operate.

Active Publication Date: 2017-02-15
SHANXI LIFEGEN
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0007] However, compared with the detection of HLA alleles, this technology has rarely been applied to the detection of HLA‐B*5801 alleles; the fundamental reason is that multiple HLA‐B alleles are highly correlated with HLA‐B*5801 Therefore, it is very difficult to design a set of primer-probe combinations suitable for the high-specificity amplification of HLA-B*5801 alleles in fluorescent PCR reactions

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  • A real-time fluorescent PCR-based method for detecting the hla‑b*5801 allele
  • A real-time fluorescent PCR-based method for detecting the hla‑b*5801 allele
  • A real-time fluorescent PCR-based method for detecting the hla‑b*5801 allele

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specific Embodiment

[0041] Specific embodiment TaqMan-MGB probe method detects HLA-B*5801 allele

[0042] 1. DNA sample extraction and dilution

[0043] Obtain ethylenediaminetetraacetic acid (EDTA) anticoagulated vacuum blood collection tubes according to conventional methods, and then use QIAamp DNA Mini Blood Kit (Qiagen, Germany) to extract DNA; use NanoDrop 2000 to measure the concentration of the extracted DNA Determination (A 260 / 280 =1.95~2.15). Using the above method, the concentrations of 104 Bouyei DNA samples were measured, and then the samples were diluted to 10 ng / μL with PCR-grade H2O.

[0044] 2. Design primers and probes

[0045] In the region where the polymorphic sites are concentrated, specific primers for HLA-B*5801 were designed using ARMS method, upstream primer F: 5'-GGGCCGGAGTATTGGGATG-3', downstream primer R: 5'-TTGGCCTCAACTGAAAATGAAAC-3', and matching The fluorescent probe probe: 5'-HEX / VIC-TCAGGGAGGCGGATCTCGGAC-MGB-3'; in addition, an internal reference primer was ...

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Abstract

The invention designs a primer-probe combination capable of amplifying HLA-B * 5801 allele with high specificity based on a TaqMan probe detecting method: Fp: 5'-AGGGGCCGGAGTATTGGGATG-3', Rp:5'-TTGGCCTCAACTGAAAATGAAAC-3', MGB probe: 5'-HEX-VI-TCAGGGAGGCGGATCTCGGAC-MGB3'; meanwhile, by virtue of primers and probes for a reference gene ACTB, a target gene and the reference gene are added into a tube to have a dual-channel fluorescence quantitative PCR reaction, and the result is analyzed by an amplification curve. The method provided by the invention is simple and convenient, flexible, fast, high in specificity, high in throughput, pollution-free, and high in resolution, and is applicable to detection of the HLA-B * 5801 allele of whole genome DNA samples in human peripheral blood and saliva.

Description

technical field [0001] The invention belongs to the field of pharmacogenomics and gene detection, in particular to a method for detecting HLA-B*5801 allele. Background technique [0002] HLA refers to human leukocyte antigen. It is encoded by a group of closely linked multiple alleles on the short arm of human chromosome 6. It consists of 3.6 million base pairs. most abundant area. It mainly includes HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ and HLA-DP. The World Health Organization HLA Factor Nomenclature Committee has named more than 5,000 related HLA alleles; the polymorphism of HLA genes determines the different HLA protein molecules expressed between individuals, and also determines the ability of different individuals to process and present the same antigen Different, this is the most basic point of immune response induction and regulation, so that the immune response of different individuals to the same antigen can show individual differences. Such individual differences...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2561/101C12Q2545/101
Inventor 康星王会娟陈融刘金辉戴鹏高陈超
Owner SHANXI LIFEGEN