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Diagnosis reagent for systemic lupus erythematosus and platform and application thereof

A diagnostic reagent and a technology for lupus erythematosus, which are applied in the field of diagnostic reagents for systemic lupus erythematosus and its platform, can solve the problems of complicated steps and are not suitable for clinical promotion and application, and achieve the effect of simple detection process and easy promotion and application.

Active Publication Date: 2020-07-28
深圳市赛尔生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It can be seen that the steps of the above-mentioned authorized patents are too complicated and not suitable for wide clinical promotion and application.

Method used

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  • Diagnosis reagent for systemic lupus erythematosus and platform and application thereof
  • Diagnosis reagent for systemic lupus erythematosus and platform and application thereof
  • Diagnosis reagent for systemic lupus erythematosus and platform and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of the diagnostic reagent of systemic lupus erythematosus

[0033] Including: (1) Reagents for extracting whole blood DNA: Thermo Scientific GeneJET Whole Blood GenomicDNA Purification Mini Kit, #K0782, Lot 00239303

[0034] Table 1

[0035]

[0036] (2) Reagents required for sulfite treatment: EZ DNA Methylation TM Kit, #D5002

[0037] Table 2

[0038]

[0039] (3) Reagents required for fluorescent quantitative PCR: LightCycler®480 High Resolution Melting Master, ref 04909631001

[0040] table 3

[0041]

[0042] Materials and instruments used in the experiment

[0043] Materials: GeneJET Genomic DNA Purification Columns pre-assembled with CollectionTubes; Collection Tubes (2mL); Pipettes and pipette tips; 1.5mL microcentrifuge tubes; Zymo-Spin TM IC Columns; Zymo-Spin TM I-96 Binding Plates (D5004); Conversion Plates w / Piercesble Cover Film;; Collection Plates; Elution Plates

[0044] Instruments: Vortex Mixer; Thermomixe...

Embodiment 2

[0045] Embodiment 2: the application of the diagnostic reagent of systemic lupus erythematosus

[0046] Step 1: Genomic DNA extraction from peripheral blood of patients with systemic lupus erythematosus (using a commercial whole blood DNA extraction kit):

[0047] (1) Take 0.5ml of whole blood into a 1.5ml centrifuge tube, then add 1ml of ice-distilled water, and mix thoroughly up and down several times; (2) Stand at room temperature for 5 minutes, then centrifuge at 800g (3000rpm) for 5 minutes; (3) Take it out and discard it (4) Add 20ul Proteinase K, vortex; (5) Add 400ul Lysis Solution, vortex or invert to mix; (6) Bath in 56℃ water for 10min, During this period, the sample was mixed upside down 3 times; (7) Take out the water bath and add 200ul absolute ethanol, mix upside down, and spot off; (8) Transfer the mixed solution to a new centrifugal adsorption column, and centrifuge at 6000g (8000rpm) for 1min; (9) ) Transfer the adsorption column to a new waste tube; (10) Ad...

Embodiment 3

[0083] Embodiment 3: The diagnostic reagent of systemic lupus erythematosus of the present invention and its platform sensitivity and specificity test

[0084] Using the above method to detect the two CGs contained in the DNA sequence SEQ ID No.1 within -2000bp upstream of the IFI44L gene transcription start site in peripheral blood of 1024 cases of SLE patients, 512 cases of healthy controls, 388 cases of RA patients and 374 cases of PSS patients The methylation level of the site, the test results showed that the average value of these two CG sites in the healthy control group was hypermethylated, and the average level of methylation at the two CG sites in the SLE patient group was significantly higher than that of the healthy control group, RA patients and SS patients. significantly lower compared to

[0085]The ROC curve was used to evaluate the sensitivity and specificity of the average methylation level of two CG sites in the diagnosis of SLE. The actual value range of th...

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Abstract

The invention relates to a diagnosis reagent of systemic lupus erythematosus and a platform and application thereof, in particular to a diagnosis reagent of systemic lupus erythematosus based on a high-resolution melting curve (HRM) method and a platform thereof. The reagent comprises a SEQ ID No. 2 upstream primer 5 '-GAAATGAAAGTAAGGAAGTTAGGAG-3' and a SEQ ID No. 3 downstream primer 5 '-AAATCCAATACTATCACTCATTCC-3'. The specific primers are adopted and matched with a high-resolution melting curve (HRM) method detection platform, so that the detection process is simpler and more convenient, andclinical popularization and application are facilitated.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a diagnostic reagent for systemic lupus erythematosus and a platform thereof, in particular to a diagnostic reagent for systemic lupus erythematosus and a platform thereof based on a high-resolution melting curve (HRM) method. Background technique [0002] Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organs and systems. The clinical manifestations are kidney, neuropsychiatric system, blood system and other multiple organ damage. Early diagnosis before vital organ involvement in SLE patients is of great significance for the prevention and treatment of SLE, improving the quality of life of patients, and improving the survival rate of patients. However, most of the current biological diagnostic markers are biochemical and immunological changes that appear after organ damage, which cannot be used for early diagnosis of organ involveme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/154C12Q2600/166C12Q2531/113C12Q2527/107C12Q2545/113
Inventor 陆前进赵明张博刘立民吴海竞
Owner 深圳市赛尔生物技术有限公司