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Method for quickly detecting human leukocyte antigen HLA-B27 gene by fluorescence quantitative Polymerase Chain Reaction (PCR) and kit thereof

A technology of HLA-B27 and human leukocyte antigen, applied in the field of biomedicine, can solve the problems of complicated operation process, false positive, false negative and so on

Inactive Publication Date: 2010-12-29
BEIJING SUOAO BIOMEDTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The operation process of this method is complicated, and it is prone to false positives and false negatives, so it is not suitable for clinical promotion.

Method used

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  • Method for quickly detecting human leukocyte antigen HLA-B27 gene by fluorescence quantitative Polymerase Chain Reaction (PCR) and kit thereof
  • Method for quickly detecting human leukocyte antigen HLA-B27 gene by fluorescence quantitative Polymerase Chain Reaction (PCR) and kit thereof
  • Method for quickly detecting human leukocyte antigen HLA-B27 gene by fluorescence quantitative Polymerase Chain Reaction (PCR) and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Collection and delivery of HLA-B27 test specimens

[0046] Draw 1-2 ml of venous blood from the subject with a sterile syringe, inject it into a sterile EDTA (or sodium citrate) anticoagulant tube, and mix immediately. Specimens can be used immediately for testing, or can be stored in -20°C or -70°C refrigerators. Specimens should not be stored for more than 24 hours at 2-8°C; at -20°C the storage period is 6 months; at -70°C they can be stored for a long time. Specimens should avoid repeated freezing and thawing, and specimens should be transported in 0°C ice cubes.

Embodiment 2

[0047] Example 2: Extraction of Genomic DNA

[0048] Shake the anticoagulated blood evenly with the DNA extraction reagent provided in this kit, transfer 500 μl to a clean 1.5ml centrifuge tube, and mark it with numbers. Add 1ml of 1× red blood cell lysate (RCLB, prepared from 10×RCLB in this kit with double distilled water at a ratio of 1:9 to make 1×RCLB), shake vigorously for 30 seconds, centrifuge at 3000g for 5 minutes, and discard the supernatant. Repeat the above steps twice until there is no obvious red precipitate. If the red precipitate is still obvious, it is necessary to repeat the above steps once more. Add 1ml of normal saline, shake vigorously for 15s, centrifuge at 10000g for 5min, and discard the supernatant. Add 50 μl of the nucleic acid extraction solution provided in the kit to the precipitation, keep the temperature at 100°C for 10 minutes, centrifuge at 13,000 g for 3 minutes, and take 4 μl of the supernatant for PCR reaction.

Embodiment 3

[0049] Embodiment 3: Applicable instruments for this detection method and kit

[0050] Applicable instruments mainly include ABI GeneAmp PCR System7700, ABI GeneAmp PCR System7500, ABI PRISM 7300, etc., as well as instruments using capillaries such as LightCycler, MJOpticon series or qualified quantitative PCR instruments.

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Abstract

The invention provides a method for quickly and accurately detecting a human leukocyte antigen HLA-B27 gene highly related to ankylosing spondylitis by using a fluorescence quantitative PCR technique (a PCR-fluorescence probe method) from a human peripheral blood sample. The method comprises the following steps: (1) collecting a sample; (2) preprocessing the sample and extracting DNA; (3) detecting the sample by a fluorescence probe in vitro amplification method; and (4) analyzing the corresponding sample after amplification reactions are finished according to the fluorescence intensity of each amplification reaction, thus judging whether a patient carries the human leukocyte antigen HLA-B27 gene highly related to the ankylosing spondylitis as clinical aided diagnosis of the ankylosing spondylitis and realizing real-time rapid and accurate detection of the human leukocyte antigen HLA-B27 gene.

Description

1. Technical field [0001] The invention relates to a method for detecting human leukocyte antigen HLA-B27 (Human Leukocyte Antigen B27) gene, in particular to a method for detecting HLA-B27 by extracting human peripheral blood genomic DNA using fluorescent quantitative PCR technology. The present invention further relates to a kit for detecting human leukocyte antigen HLA-B27 gene. The invention belongs to the field of biomedicine. 2. Background technology [0002] Human leukocyte antigen (HLA) is the expression product of human major histocompatibility complex (Major Histocompatibility Complex, MHC). The HLA gene is located on the short arm of the sixth pair of chromosomes, about 4000kb. According to different functions and products, HLA genes are divided into three categories: classic HLA genes, immune function-related genes, and immune-related genes. HLA genes include A, B, C, D, DR, DQ, DP and other loci, and there are different loci in these types of loci, which is o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 史成军任美峰徐贵峰刘健翊
Owner BEIJING SUOAO BIOMEDTECH
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