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Fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes

A HLA-B, qualitative detection technology, applied in the direction of fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., to achieve the effect of accurate qualitative, simple steps, and reduce the risk of PCR product contamination

Active Publication Date: 2014-03-12
CHANGSHA 3G BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no product on the market that uses fluorescent PCR technology for HLA-B genotyping

Method used

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  • Fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes
  • Fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes
  • Fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of kit

[0033] 1. Design and synthesis of primers and probes

[0034]Using Primer Express 2.0, for exon 2 and exon 3 of HLA-B*1502 and the intron region in the middle, studies have shown that the current HLA-B genotyping is mainly concentrated in this region (Hughes AL, Nei M .Pattern of nucleotide substitution at MHC class I loci reveals overdominant selection. Nature, 1988, 335(6186): 167-170; Hughes AL, Yeager M. Natural selection at major histo-compatibility complex loci of vertebrats. Annu Rev Genet, 1998 , 32: 415-435; Gao X, Nelson GW, Karacki P, Martin MP, Phair J, Kaslow R, Goedert JJ, Buchbinder S, Hoots K, Vlahov D, O′Brien SJ, Carrington M. Effect of a single amino acid change in MHC class I molecules on the rate of progression-sion to AIDS.N Engl J Med, 2001, 344(22): 1668-1675; Xu Junping, Deng Zhihui, Zou Hongyan, etc., Chinese Han individuals HLA-A, - Determination of the full-length sequence of the B gene and the polymor...

Embodiment 2

[0068] Embodiment 2: the use of kit

[0069] 1. Sample detection

[0070] The positive control and negative control were respectively taken as DNA templates, and added to UNG enzyme, Taq polymerase, and fluorescent quantitative reaction solution containing specific PCR primers and specific fluorescent probes to form a PCR reaction system. In the positive control substance, the HLA-B*1502 plasmid standard substance (5.0 × 10 6 copy / microliter), as a standard for qualitative determination of samples.

[0071] The main components of the system are as follows:

[0072]

[0073] 2. Reaction procedure

[0074] Set the fluorescence detection channel for collecting FAM and VIC fluorescence signals, put the reaction tube into the fluorescence PCR instrument (ABI7500) to start amplification, the reaction procedure is as follows:

[0075] Table 3. PCR reaction program

[0076]

[0077] 3. Result judgment

[0078] The Ct value (cycle number) in the baseline range is 6-15 or au...

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PUM

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Abstract

The invention provides a fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes, and belongs to the field of in-vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, PCR genotyping primers and a fluorescence probe; the PCR genotyping primer sequence is as shown in SEQ ID NO: 3-4 and / or SEQ ID NO: 6-7. The kit provided in the invention has high sensitivity and good specificity, and can monitor reaction process in real time and the reaction time is short; in addition, closed tube operation is performed, and subsequent treatment is not needed, which can maximally avoid the pollution of a reaction product, thus being capable of replacing traditional cell detection.

Description

technical field [0001] The invention relates to the field of in vitro nucleic acid detection, in particular to a fluorescent polymerase chain reaction (PCR) kit for distinguishing 1502 gene subtypes in HLA-B genes in clinical samples. Background technique [0002] The HLA (human leukocyte antigen, human leukocyte antigen) system is the most complex polymorphic system known to the human body. Since the discovery of the first HLA antigen (Jean Dausset) in 1958, until the 1970s, HLA has become an important emerging research field in the disciplines of immunogenetics, immunobiology and biochemistry. HLA is a highly polymorphic alloantigen, and its chemical essence is a kind of glycoprotein. HLA is divided into class I antigens and class II antigens according to their distribution and function. HLA class I antigens are encoded by HLA-A, B, and C sites; HLA class II antigens are controlled by the HLA-D region (including 5 subregions). The above genes (revised by the WHO Nomencl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 张璇
Owner CHANGSHA 3G BIOTECH
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