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HLA-B27 (Human Leukocyte Antigen-B27) allele detection kit and application thereof

An HLA-B27, 1. HLA-B27 technology, applied in the field of gene detection kits, can solve the problems of complex qPCR reaction system, unstable results, etc. Effect

Active Publication Date: 2018-09-04
广东辉锦创兴生物医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the richness of the HLA-B27 allele sequence, the existing qPCR method can only use multiple primers and probes to cover the entire sequence of the HLA-B27 allele, resulting in a complex qPCR reaction system and unstable results

Method used

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  • HLA-B27 (Human Leukocyte Antigen-B27) allele detection kit and application thereof
  • HLA-B27 (Human Leukocyte Antigen-B27) allele detection kit and application thereof
  • HLA-B27 (Human Leukocyte Antigen-B27) allele detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Sequence Alignment Analysis of Existing HLA-B27 Genotypes

[0023] In order to overcome the defects in the above-mentioned qPCR method, the present invention provides a probe with degenerate bases, and can detect the HLA-B27 genotype in the largest range under the condition of ensuring the lowest degeneracy. After analyzing the known HLA-B27 sequence, find out its relatively conservative sequence, and analyze the differential base sequence, such as figure 1 shown.

[0024] Considering that in the Chinese population, 2704 and 2705 account for the highest proportion and are positively correlated with the occurrence of AS, the primers and probes for the detection of HLA-B27 in the present invention, and the primers and probes for the ActB gene that have been used as internal references are shown in Table 1.

[0025] Table 1. Primer and probe sequences of HLA-B27 gene and internal reference gene ActB

[0026]

[0027]

[0028]Note: The degenerate base r o...

Embodiment 2

[0029] Example 2. Sample HLA-B27 Positive / Negative Detection

[0030] The reaction was carried out through the following fluorescent quantitative PCR reaction system. The qPCR reaction system used the product of Beijing Kangwei Century Biotechnology Company, as shown in Table 2, and the reaction conditions were shown in Table 3.

[0031] Table 2. Reaction system for detecting HLA-B27 alleles by qPCR

[0032]

[0033] Table 3. qPCR reaction conditions

[0034]

[0035] On the fluorescent quantitative PCR instrument ABI7500, the fluorescent quantitative reaction curves for detecting HLA-B27 positive and negative can be found in figure 2 a and 2b.

[0036] 20 cases of HLA-B27 positive and 20 cases of HLA-B27 negative samples confirmed by Sanger sequencing method were taken and tested by the method of the present invention, and the positive and negative results were 100% coincident.

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Abstract

The invention relates to an HLA-B27 (Human Leukocyte Antigen-B27) allele detection kit and the application thereof. The kit comprises a qPCR (Quantitative Polymerase Chain Reaction) primer and a probefor detecting HLA-B27 allele. By adopting the primer and the probe of the kit, the defects that a qPCR reaction system is complex and results are not stable since sequences of HLA-B27 allele can be completely covered by using a plurality of primers and probes in a conventional qPCR method, can be overcome. The kit provided by the invention is simple to use, high in accuracy, monitorable in process, rapid in result, free of electrophoresis process and beneficial to clinical popularization and application.

Description

technical field [0001] The invention relates to the field of molecular diagnosis, in particular to an HLA-B27 allele detection kit and application thereof. Background technique [0002] Ankylosing spondylitis (AS) is a chronic autoimmune disease that mainly affects the spine, axial bones, and large joints of the extremities, and is characterized by fibrosis of the intervertebral disc annulus and its connective tissue, bone fusion, and joint ankylosis. . Those with spondylitis as the main lesion are called primary AS, while those accompanied by reactive arthritis, psoriasis, and inflammatory bowel disease are called secondary AS. The hallmark feature of this disease is that it begins with sacroiliitis and gradually spreads to the spine and paraspinal soft tissues. AS is often hereditary, can cause teratogenicity, and has no significant impact on the patient's life expectancy. AS is a sporadic disease, and its incidence varies slightly in race and region. It is about 0.05%...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/107C12Q2545/101C12Q2545/113
Inventor 胡锦张凤笑
Owner 广东辉锦创兴生物医学科技有限公司
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