Multiplex real-time fluorescent PCR (polymerase chain reaction) method for detecting HLA-B*15:02 alleles

An HLA-B, multiple fluorescence technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of few genotypes, complicated operations, false positives, etc., to achieve low cost and improve sensitivity , the effect of high sensitivity

Active Publication Date: 2015-08-12
SHANXI LIFEGEN
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are kits for detecting HLA-B*15:02 based on the RT-PCR method, the kits cover fewer genotypes, are cumbersome to operate, require multi-tube reactions, and are prone to false positives and contamination, which is far from being satisfactory. Rapid, simple and highly specific detection of HLA-B*15:02 gene needs

Method used

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  • Multiplex real-time fluorescent PCR (polymerase chain reaction) method for detecting HLA-B*15:02 alleles
  • Multiplex real-time fluorescent PCR (polymerase chain reaction) method for detecting HLA-B*15:02 alleles
  • Multiplex real-time fluorescent PCR (polymerase chain reaction) method for detecting HLA-B*15:02 alleles

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Embodiment

[0040] Example TaqMan probe method for detection of HLA-B*15:02 alleles

[0041] 1. DNA sample extraction and dilution

[0042] After collecting venous blood in vacuum blood collection tubes anticoagulated with ethylenediaminetetraacetic acid (EDTA) according to conventional methods, DNA was extracted using the QIAamp DNA Mini Blood Kit (Qiagen, Germany) kit; the extracted DNA was concentrated using NanoDrop 2000 Determination (A 260 / 280 =1.95~2.15). Using the above method, respectively measure the concentration of 200 cases of Bouyei DNA samples, and then use PCR-grade H 2 O Dilute the sample to 10 ng / μL.

[0043] 2. Design primers and probes

[0044] In the region where the polymorphic sites are concentrated, use the ARMS method to design specific primers for HLA-B*15:02:

[0045] The first upstream primer F1: 5'-GACCGGACCACACAGATCCC-3';

[0046] The first downstream primer R1: 5'-ATGGGGAGTCGTGACCTG-3';

[0047] The first fluorescent probe probe1: 5’-HEX-ACCTGCGCGGCTA...

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Abstract

The invention discloses a primer-probe combination of two pairs of high specific amplification HLA-B*15:02 alleles designed on the basis of using a TaqMan probe detection method. On the basis, by using a primer and probe of a reference gene beta-Actin, specific primers and probes of two pairs of target genes and the primer and probe of the reference gene are added into a same pipe to carry out a multiplex fluorescent PCR, and then results are analyzed by using a fluorescent amplification curve. The method disclosed by the invention has the characteristics of high specificity, flexibility, rapidness, high flux, no pollution, high resolution, capability of carrying out real-time monitoring on a reaction process, and the like, and can be applied to the detection of HLA-B*15:02 alleles of whole-genome DNA samples in human peripheral blood and saliva.

Description

technical field [0001] The invention belongs to the field of pharmacogenetic diagnosis, and in particular relates to a method for detecting HLA-B*15:02 alleles and the design of primers and probes thereof. Background technique [0002] HLA refers to human leukocyte antigen, which is encoded by a group of closely linked multiple alleles on the short arm of human chromosome 6, including more than 100 gene loci, and more than 9,000 alleles have been found, with a total length of 3600kb, which is currently known The gene density is the highest in the human chromosome, and it is also the region with the most polymorphism. The number of HLA alleles named by the HLA Factor Nomenclature Committee of the World Health Organization has reached more than 5,000; in view of the high polymorphism and complexity of the HLA system, the detection method of HLA alleles is different from the detection of ordinary polymorphic sites . [0003] Carbamazepine is a tricyclic anticonvulsant drug, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王会娟康星陈融刘正斌韩敏周少荷陈超
Owner SHANXI LIFEGEN
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