Primer, probe, fluorescence PCR (Polymerase Chain Reaction) kit and method for detecting HLA (Human Leucocyte Antigen)-B*5801 genes

A technology for HLA-B and detection primers, applied in the field of biomedical clinical molecular detection, can solve the problems of long cycle, high price, cumbersome data analysis, etc., and achieve the effect of low cost, extensive promotion value, and guaranteeing authenticity and accuracy.

Inactive Publication Date: 2015-03-11
SUZHOU KUANGYUAN MOLECULAR BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the disadvantages of the prior art, such as high price, long period, low efficiency, cumbersome data analysis, etc., when detecting the HLA-B*5801 allele, and provides a HLA-B*5801 based on the Taqman real-time fluorescent quantitative PCR technology platform. Primers, fluorescent probes, kit and detection method for allele detection, used for detection of human HLA-B*5801 allele

Method used

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  • Primer, probe, fluorescence PCR (Polymerase Chain Reaction) kit and method for detecting HLA (Human Leucocyte Antigen)-B*5801 genes
  • Primer, probe, fluorescence PCR (Polymerase Chain Reaction) kit and method for detecting HLA (Human Leucocyte Antigen)-B*5801 genes
  • Primer, probe, fluorescence PCR (Polymerase Chain Reaction) kit and method for detecting HLA (Human Leucocyte Antigen)-B*5801 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1. Design of primers and probes:

[0080] According to the reference sequence of HLA-B gene (NG_023187) published by the nucleic acid sequence database GeneBank of the National Center for Biotechnology Information of the United States (NG_023187) and the information of HLA-B*5801 (HLA00386) published by the British IMG / HLA database, Primer Express of ABI Company was used. 3.0 software designed primers and probes to detect HLA-B*5801 and other genes respectively. Primers and probes, as follows:

[0081] Forward primer:

[0082] HLA-B*5801F: 5’-CACGGAACATGAAGGCCTCC-3’

[0083] Reverse primer:

[0084] HLA-B*5801 R: 5’-CGGAGGAGGCGCCCGTAG-3’

[0085] Fluorescent Probes:

[0086] TM-HLA-B*5801: 5’Fam-CCCAGCGCGTTTACCCGGTTT-3’BHQ1

[0087]In order to monitor the effectiveness of the reaction system, the internal control primers and probes are added to the detection system. The present invention selects a segment of the human conserved gene GAPDH (the reference se...

Embodiment 2

[0093] Example 2. Preparation of primers

[0094] The designed primers and probe sequences are synthesized by a synthesis company. Generally, automatic chemical synthesis is used by instruments, and a synthesis inspection report is required.

Embodiment 3

[0095] Example 3: Preparation of a kit for fluorescent PCR detection of human HLA-B*5801 allele

[0096] A reaction solution for detecting human HLA-B*5801 alleles was prepared, wherein the PCR reaction solution contained the necessary buffer solution, magnesium ions, dNTPs and other substances for the PCR reaction, as well as detection primers and probes and internal control primers and probes. The above-mentioned specific detection PCR reaction solution component concentrations and the sequence information of the included primers and probes are as follows:

[0097] Table 1. PCR reaction solution components

[0098] Reaction liquid components concentration buffer 10× Magnesium ions 15mM dNTP (A / T / G / C) 20mM each Upstream detection primers 2μM Downstream detection primers 2μM detection signal probe 2μM Upstream internal control primer 1μM Downstream internal control primer 1μM Internal control signal probe ...

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Abstract

The invention provides a primer, a probe, a fluorescence PCR (Polymerase Chain Reaction) kit and a method for detecting HLA (Human Leucocyte Antigen)-B*5801 alleles. Human leucocyte antigen (HLA)-B*5801 alleles are detected on a real-time fluorescent quantitative PCR technical platform according to the principle of 'allele specific PCR'.

Description

technical field [0001] The invention belongs to the field of biomedical clinical molecular detection, in particular to primers, probes, fluorescent PCR kits and detection methods for HLA-B*5801 allele detection. Background technique [0002] Human leukocyte antigen (HLA) is a group of genes located on chromosome 6. It is the gene complex with the highest allelic polymorphism among known genes so far. More than 2,700 alleles have been found in the world, although how many of them are Alleles are rare. HLA gene is the most complex genetic polymorphism system in humans, and its polymorphism distribution has obvious ethnic characteristics. So far, studies in Han Chinese from mainland China, Hong Kong, Taiwan, and Thailand have shown a strong association between the HLA-B*5801 allele and severe skin adverse reactions to the drug allopurinol. In European and Japanese races, the correlation is relatively low. [0003] Allopurinol is an inhibitor of hypoxanthine oxidase, which ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/166
Inventor 王进何晓辉赵小龙吴梦华唐明慧吴子侠
Owner SUZHOU KUANGYUAN MOLECULAR BIOTECH
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