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Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene

A real-time fluorescence and reagent kit technology, applied in the field of biotechnology detection, can solve the problems of high cost, long qualitative and quantitative cycles, and not suitable for large-scale use

Inactive Publication Date: 2015-07-15
李彩香
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, in the detection method of APRIL, the direct detection and quantitative technology of antibodies is still widely used; however, for the current personalized treatment of patients and the detection of disease development, this technology has a long qualitative and quantitative cycle and high cost, and is not suitable for large-scale usage of

Method used

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  • Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene
  • Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene
  • Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1) Centrifuge the sample to be tested and the negative control blood, extract the RNA of the lower blood cells, reverse transcribe the cDNA solution, and store it at low temperature for later use; the test samples are taken from the blood of patients with systemic lupus erythematosus (1, 2, 3) and primary Blood from patients with biliary cirrhosis (4); normal human serum (5, 6) as negative controls.

[0028] 2) Reaction system: Take 2ul of the DNA solution obtained in step 1), mix it with 14ul of the detection solution in the above kit, and add sterile ultrapure water to a reaction volume of 20ul; add it to a real-time fluorescence reaction tube, and carry out Centrifuge; at the same time, use sterile water as a blank control

[0029] 3) Perform real-time fluorescent PCR detection on the reaction system in step 2) according to the following conditions:

[0030] Pre-denaturation at 95°C / 100s, 1 cycle; denaturation at 95°C / 30s, extension at 66°C / 120s, 40 cycles.

[0031...

Embodiment 2

[0034] Utilize ELISA to carry out the concentration of serum APRIL in the detection sample, adopt the microplate reader to carry out the analysis of the OD value obtained by detection; And real-time fluorescent PCR detection result is consistent; The APRIL antibody used is purchased from Santa Cruz company, and article number is APRIL (F- 5).

[0035] The detection result of ELISA is consistent with the expression trend of APRIL obtained by real-time fluorescent PCR.

[0036]

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Abstract

The invention provides a primer for real-time fluorescence PCR (polymerase chain reaction) detection of an APRIL (a proliferation inducing ligand) gene. The forward primer (SEQ ID NO.1) is 5-GTGATTTAAGAGGACT-3, and the reverse primer (SEQ ID NO.2) is 5-TGGAGAGGGTGCGAAC-3. The invention also provides a kit and a method for real-time fluorescence PCR detection of the APRIL gene through the primer. The invention adopts a SYBR Green I real-time fluorescence PCR detection method for indentifying the APRIL gene, has detection instantaneity and high efficiency, and can be used for detecting the expression of early APRIL gene.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to primers and kits for real-time fluorescent PCR detection of APRIL gene. Background technique [0002] A proliferation-inducing ligand (a proliferation inducing ligand, APRIL), and BAFF (B-cell-activating factor) belong to the tumor necrosis factor superfamily members, mainly secreted by DC, macrophages, T and B cells. The function of APRIL for immune regulation is less defined. Recent studies have shown that APRIL may act as a co-stimulator of naive B and T cells and induce peripheral blood B cells to produce IgM [G.Yu, T.Boone, J.Delaney et al, Nat Immunol, 2000, 1 (3): 252-256 ; J.V. Stein, M. Lopez-Fraga, F.A., J Clin Invest, 2002, 109(12): 1587-1598], affecting thymus-dependent B cell responses. Like BAFF, APRIL promotes B cell proliferation and increases the number of activated T cells. [0003] APRIL can not only bind to the co-receptors BCMA and TACI of BAFF, but ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/158
Inventor 李彩香刘树霞朱文斌
Owner 李彩香
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