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A biomarker for early diagnosis of ankylosing spondylitis and its application in kit

A technology for ankylosing spondylitis and auxiliary diagnosis, applied in biochemical equipment and methods, microbiological determination/testing, DNA/RNA fragments, etc.

Active Publication Date: 2021-07-06
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In view of the fact that there is no report on the application of the CMYA5 gene SNP site in the diagnosis of ankylosing spondylitis, if the SNP susceptible to ankylosing spondylitis can be screened out as a biomarker, and a corresponding diagnostic kit can be developed, it will be a strong impetus. The current status of early diagnosis of ankylosing spondylitis in my country, and to open up new ways for drug screening, drug efficacy evaluation and targeted therapy

Method used

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  • A biomarker for early diagnosis of ankylosing spondylitis and its application in kit
  • A biomarker for early diagnosis of ankylosing spondylitis and its application in kit
  • A biomarker for early diagnosis of ankylosing spondylitis and its application in kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Blood Sample Collection and Genomic DNA Extraction

[0028] From January 2017 to October 2017, the inventor selected cases according to the revised diagnostic criteria in New York in 1984. A total of 6 unrelated AS patients from Peking Union Medical College Hospital and 6 healthy control volunteers were selected to collect blood samples. . All clinical samples in this study were informed to the patients and approved by the Hospital Ethics Committee.

[0029] Methods of DNA extraction from peripheral blood:

[0030] The specific steps are:

[0031] 1. Add hemolysis reagent (i.e., lysate, 40 parts) to the peripheral blood stored in a 2mL cryopreservation tube. The volume of the solution was adjusted to 2000mL, the same below), and it was completely transferred after inverting to mix well.

[0032] 2. Removal of red blood cells: Fill the 5mL centrifuge tube to 4mL with hemolysis reagent, mix by inverting, centrifuge at 4000rpm for 10 minutes, and discard the ...

Embodiment 2

[0038] Example 2 Whole Exome Detection of SNP in Peripheral Blood DNA

[0039] The two groups of people in Example 1 were tested by whole exome sequencing to obtain relevant results.

[0040]1. Exon capture: Agilent SureSelectHumanAll Exon 70M (V4+UTRs) exome liquid phase capture chip was used for hybrid capture, with an average capture efficiency of 70%. The general process is to incubate the interrupted genomic DNA with the SureSelect bait, fish out the RNA bait-DNA hybrid through streptavidin-labeled magnetic beads, elute the magnetic beads, and degrade the RNA bait , enrich the target region, and then perform high-throughput sequencing.

[0041] 2. Exome library construction: For DNA samples that pass the quality inspection, the exome library is constructed using the Illumina standard DNA true-seq library construction process. The library construction process is briefly described as follows:

[0042] (1) Take 5 μg of genomic DNA, and use Bioruptor to perform random mech...

Embodiment 3

[0053] Example 3 Sanger sequencing verification

[0054] The present invention utilizes the Sanger sequencing method to verify the rs3828611 site.

[0055] Sanger sequencing was performed on 6 unrelated AS patients in Example 1 and 6 healthy control volunteers.

[0056] 1. DNA extraction

[0057] The step of extracting sample DNA is the same as in Example 1;

[0058] 2. Primer design and PCR reaction

[0059] Design of PCR primers: The primers were designed and synthesized by Shanghai Sangon, and the primers are shown in Table 2.

[0060] Table 2 Primer Sequence

[0061]

[0062] The PCR amplification system is shown in Table 3; the PCR amplification program is shown in Table 4.

[0063] Table 3 reaction system

[0064]

[0065]

[0066] Table 4 reaction system

[0067]

[0068] 3. Sequencing

[0069] After PCR amplification, take 5 μL of the amplified product, electrophoresis on 1% agarose gel, electrophoresis for 30 minutes, staining for 20 minutes, and t...

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Abstract

The invention discloses a biomarker for early diagnosis of ankylosing spondylitis. The marker is CMYA5 gene carrying SNP site rs3828611, and the rs3828611 site is related to the occurrence of ankylosing spondylitis. The invention can make the diagnosis of ankylosing spondylitis more convenient and easy through the development and application of SNP genotype diagnostic reagents and diagnostic kits, and can help clinicians quickly and accurately grasp the patient's condition, lay the foundation for clinical treatment effect evaluation, and lay a foundation for the discovery of ankylosing spondylitis. New small molecule drug targets with potential therapeutic value to help.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biomarker for early diagnosis of ankylosing spondylitis and its application in a kit. Background technique [0002] Ankylosing spondylitis (AS) is an autoimmune disease that mostly occurs in young adults aged 16-40, with a male to female ratio of about 4 to 10:1. Lesions often first occur in the sacroiliac joints, and a small number of severe cases show rigidity of the entire spine. In addition, some patients have different degrees of extra-spinal lesions such as hip joints, eyes, lungs, cardiovascular, and kidneys. The incidence of AS in the Caucasian population is about 1% to 3%, and the prevalence of AS in my country is about 0.2-0.6%, of which more than 60% of the patients are accompanied by hip joint involvement, resulting in more than 20% of AS patients with disability, inflammation It mainly involves the bony attachment points of joint capsules, tendons and ligaments, resul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 范彧陈俊叶伟亮
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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