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Method for rapidly detecting human leucocyte antigen B27 (HLA-B27) and kit thereof

A white blood cell antigen detection technology, applied in the field of clinical medical biological detection, can solve the problems of unfavorable clinical specimen detection, cumbersome operation, and electrophoresis of products, and achieve the effect of high cost, high sensitivity and low cost

Active Publication Date: 2012-05-09
浙江夸克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional serological methods, because it is difficult to obtain high-quality monovalent antiserum, a single serum is easy to miss or false positive, and it is often difficult to judge the result when some sera are positive when using multiple sera
The ELISA method is cumbersome and time-consuming, which is not conducive to the detection of large quantities of clinical specimens; flow cytometry detection has high sensitivity and specificity, but requires the configuration of flow cytometry, high hardware requirements, and the detection results are affected by the quality of specimens, antibodies Titer effect, prone to false positive or false negative
SSP-PCR has good sensitivity and specificity, but the product needs electrophoresis, the operation is cumbersome, and there are certain false positives or false negatives

Method used

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  • Method for rapidly detecting human leucocyte antigen B27 (HLA-B27) and kit thereof
  • Method for rapidly detecting human leucocyte antigen B27 (HLA-B27) and kit thereof
  • Method for rapidly detecting human leucocyte antigen B27 (HLA-B27) and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Primer and probe sequences for detecting HLA-B27.

[0045] Entrust Shanghai Yingjun Biotechnology Co., Ltd. to synthesize:

[0046] SEQ ID NO: 1 5'-GGGTCTCACACCCTCCAGAAT-3'

[0047] SEQ ID NO: 2 5'-CGGCGGTCCAGGAGCT-3'

[0048] SEQ ID NO: 3 5'-FAM-CGTTCAGGGCGATGTAATCCTTGC-TAMRA-3'

[0049] SEQ ID NO: 4 5'-ATTCTGGAGGGTGTGAGACCC-3'

[0050] SEQ ID NO: 5 5'-AGCTCCTGGACCGCCG-3'

[0051] SEQ ID NO: 6 5'-FAM-GCAAGGATTACATCGCCCTGAACG-TAMRA-3'

Embodiment 2

[0052] Embodiment 2: the preparation method of kit.

[0053] (1) DNA extraction solution A:

[0054] 0.32M sucrose (purchased from Sigma, USA), 10mM Tris-HCl pH 7.5 (purchased from Sigma, USA), 5mM MgCl 2 (purchased from Sigma, USA), 0.1% (v / v) Triton X-100 (purchased from Sigma, USA);

[0055] (2) DNA extraction solution B:

[0056] 30mM Tricine pH 8.5 (purchased from Sigma, USA), 210mM ​​sodium octanoate (purchased from Sigma, USA), 2.5% (m / v) Chelex-100 (purchased from Bio-rad, USA);

[0057] (3) PCR reaction solution:

[0058] 0.2 μ M primer pair SEQ ID NO: 1 and 2 (or SEQ ID NO: 4 and 5), 0.04 μ M fluorescent probe SEQ ID NO: 3 (or SEQ ID NO: 6), 30 mM pH 8.9 Tricine (purchased from Sigma, USA company), 0.05% (v / v) Tween-20 (purchased from Sigma, USA), 0.01% (m / v) gelatin (purchased from Sigma, USA), 0.03% (m / v) BSA (purchased from Sigma, USA company), 6.5mM MgCl 2 (purchased from Sigma, USA);

[0059] (4) 5U / μl Taq DNA polymerase (purchased from Fermentas, USA), s...

Embodiment 3

[0062] Embodiment 3: detection method.

[0063] Instrument: Roche Fluorescent quantitative PCR detector, BECKMAN 22R desktop micro-refrigerated centrifuge, Taicang Hualida Laboratory Equipment Company WH-866 vortex oscillator.

[0064] (1) Extracting human genomic DNA from peripheral blood, specifically including the following steps:

[0065] (1a) Take 3ml of venous blood with a sterile injection needle into a closed sterile glass tube containing EDTA-K2, mix gently, centrifuge at 3000rpm for 5min, absorb 300ul of white blood cell layer in the middle and add 1ml of DNA extraction solution A to 1.5ml In a centrifuge tube, mix well and incubate at room temperature for 3-10 minutes (during this period, turn the centrifuge tube 10 times), so that the red blood cells are destroyed and the liquid becomes transparent.

[0066] (1b) Centrifuge at 13000rpm for 30s, carefully aspirate and discard the supernatant, leaving a visible white blood cell sediment layer. Add 500ul of DNA ...

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Abstract

The invention relates to a method for rapidly detecting a human leucocyte antigen B27 (HLA-B27) and an in-vitro diagnosis kit, which belong to the technical field of medical biology. In the kit, a pair of HLA-B27 specific primers and a specific fluorescent probe are adopted for rapidly detecting the HLA-B27. In the kit, the pair of HLA-B27 specific primer and the specific fluorescent probe can beused for rapidly and accurately detecting HLA-B27 genes in samples such as human peripheral blood and the like, and the kit has the advantages of high specificity, high sensitivity, time saving, labor saving, low cost, high flux and the like, and can be applied to auxiliary diagnosis of multiple spondyloarthropathies which severely endanger human health such as clinical ankylosing spondylitis, Reiter syndrome, psoriatic arthritis, ulcerative colitis accompanying arthropathy, acute anterior tract uveal, and the like.

Description

technical field [0001] The invention belongs to the technical field of clinical medical biological detection, in particular to a rapid human leukocyte antigen B27 (HLA-B27) kit and detection method. Background technique [0002] In tissue or organ transplantation, compatibility (non-rejection) or incompatibility (rejection) between donor and recipient is determined by their tissue specificity. This kind of tissue antigen representing individual specificity is called histocompatibility antigen, and a set of such antigen system is called major histocompatibility system (major histocompatibility system, MHS). The gene group encoding MHS is called the major histocompatibility complex. MHC refers to a group of closely linked gene groups on a certain chromosome. [0003] Human MHC is called human leukocyte antigen (human leukocyte antigen, HLA). When HLA is used as an antigen, it is called HLA antigen system; when HLA is used as a gene, it is called HLA complex. It is located on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 商纯尔张伟林
Owner 浙江夸克生物科技有限公司
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