CN103484559B - Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene - Google Patents
Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene Download PDFInfo
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- CN103484559B CN103484559B CN201310493575.7A CN201310493575A CN103484559B CN 103484559 B CN103484559 B CN 103484559B CN 201310493575 A CN201310493575 A CN 201310493575A CN 103484559 B CN103484559 B CN 103484559B
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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
The invention belongs to field of biological technology detection, especially relate to APRIL gene real-time fluorescent PCR testing primer and test kit.
Background technology
A kind of proliferative induction part (a proliferation inducing ligand, APRIL), and BAFF(B-cell-activating factor) belong to tumor necrosis factor superfamily member, primarily of DC, scavenger cell, T and B cell secretion.APRIL to immunoregulatory function define less.Nearest research shows that APRIL may as the costimulating factor of initial B and T cell, inducing peripheral blood B cell produce IgM [G.Yu, T.Boone, J.Delaneyet al, Nat Immunol, 2000,1(3): 252-256; J.V.Stein, M.Lopez-Fraga, F.A., J Clin Invest, 2002,109(12): 1587-1598], affect the response of thymus dependent B cell.Identical with BAFF, APRIL promotes B cell proliferation and increases the quantity of activated T cell.
APRIL not only can in conjunction with co-receptor BCMA and TACI of BAFF, so and APRIL can in conjunction with acceptor--heparan sulfate proteoglycan (heparan sulfate proteoglycans, HSPG) alone.APRIL can in conjunction with the HSPG of tumor cells expression, participates in maintenance and the growth of tumour, utilizes heparin (Heparin) can suppress the combination of APRIL and HSPG, reduces its growth promoting function to tumour; More rapid in the tumor cell ratio control group propagation of nude mice injection ARPIL transfection, suppress APRIL to have obvious restraining effect to tumor growth.At primary biliary cirrhosis (Primary biliary cirrhosis, PBC), a kind of autoimmune liver disease, the serum-concentration of patient BAFF and APRIL obviously increases [P.Rennert, P.Schneider, T.G.Cacheroet al., J Exp Med, 2000,192(11): 1677-1684].ARPIL also becomes the emerging drug target for the treatment of autoimmune disorder and tumour.
At present, in the detection method of APRIL, still generally adopt antibody direct-detection quantitative technique; But for personalized treatment and the disease progression context of detection of current patient, this technology quantitative and qualitative analysis cycle is longer, cost is comparatively large, is not suitable for large-scale use.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, the object of the invention is to provide a kind of APRIL gene real-time fluorescent PCR testing primer and test kit.
The technical solution adopted in the present invention is as follows:
The primer that APRIL gene PCR detects,
Forward primer: 5-GTGATTTAAGAGGACT-3;
Reverse primer: 5-TGGAGAGGGTGCGAAC-3;
Primer pair is utilized to carry out the test kit detected:
Test kit comprises a kind of detection solution, and it detects solution and contains containing SYBR Premix Ex Taq (2 ×), forward primer 10 μMs, reverse primer 10 μMs, Rox Dye(50 ×);
The step utilizing this test kit to carry out detecting is as follows:
1) RNA of detected sample is extracted, and reverse transcription cDNA solution.
2) reaction system: the DNA solution getting gained in 2ul step 1), and mix with the detection solution 14ul in mentioned reagent box, and add aseptic ultrapure water to reaction volume 20ul; Join in real-time fluorescence reaction tubes, carry out centrifugal;
3) by step 2) reaction system carry out real-time PCR detection according to following condition:
Denaturation 95 DEG C/100s, 1 circulation; Sex change 95 DEG C/30s, extends 66 DEG C/120s, 40 circulations.
The present invention adopts SYBR Green I real-time fluorescence PCR detection method, APRIL gene is identified, have and detect real-time and high efficiency, and can detect early stage APRIL genetic expression, extracellular region RNA for APRIL genetic expression is that detected object carries out design primer, detect the genetic expression of early stage APRIL secreted form, for carly fruit drop autoimmune disorder, can carry out in individual character Diagnosis and Treat process to patient in addition, the detection of the expression conditions of APRIL, and judge autoimmune disorder patient recurrence and increase the weight of the state of an illness possibility and for APRIL target dependency in tumour patient.
Accompanying drawing explanation
Fig. 1 is the detection sample real-time PCR detection curve of embodiment 1;
Fig. 2 is detection sample and the contrast real-time PCR detection comparison diagram of embodiment 1;
Fig. 3 is that ELISA comparison figure is carried out in the detection sample of embodiment 2 and contrast.
Embodiment
According to embodiment and accompanying drawing, the present patent application is described further.
The primer that design APRIL gene PCR detects, the extracellular region cDNA according to coding APRIL designs
Forward primer: 5-GTGATTTAAGAGGACT-3
Reverse primer: 5-TGGAGAGGGTGCGAAC-3;
Test kit comprises a kind of detection solution in addition, and it detects solution and contains containing SYBR Premix Ex Taq (2 ×), forward primer 10 μMs, reverse primer 10 μMs, Rox Dye(50 ×);
Embodiment 1
1) centrifugal detected sample and negative control blood, extracts the RNA of lower floor's hemocyte, and reverse transcription cDNA solution, cryopreservation is for subsequent use; Wherein detect sample and take from lupus erythematosus patients blood (1,2,3) and primary biliary cirrhosis patient blood (4); Normal human serum (5,6) is as negative control.
2) reaction system: the DNA solution getting gained in 2ul step 1), and mix with the detection solution 14ul in mentioned reagent box, and add aseptic ultrapure water to reaction volume 20ul; Join in real-time fluorescence reaction tubes, carry out centrifugal; Simultaneously using sterilized water as blank
3) by step 2) reaction system carry out real-time PCR detection according to following condition:
Denaturation 95 DEG C/100s, 1 circulation; Sex change 95 DEG C/30s, extends 66 DEG C/120s, 40 circulations.
Accompanying drawing 1 is the real-time fluorescent PCR amplification curve of the present embodiment, and wherein 1-3 is lupus erythematosus patients sample, and 4 is primary biliary cirrhosis patient samples; 5,6 is negative control.Wherein sample 1 and 3 curve is respectively higher than 2 and 4, and the curve of sample 2 is higher than sample 3; And show good specificity.
Accompanying drawing 2 compares for the curve fluorescence carrying out detecting sample and control sample; Sample 1-4 is apparently higher than negative control; Wherein
* *, be P<0.01.
Embodiment 2
ELISA is utilized to carry out, to the concentration of serum APRIL in detection sample, adopting microplate reader to carry out detection gained OD value and analyzing; And real-time PCR detection result compatibility; APRIL antibody used is purchased from being Santa Cruz company, and article No. is APRIL (F-5).
Detected result and the APRIL utilizing real-time fluorescence PCR to draw of ELISA express trend and conform to.
Claims (2)
- The primer of 1.APRIL gene real-time PCR detection, is characterized in that, the sequence of forward primer is as shown in SEQ ID NO.1, and the sequence of reverse primer is as shown in SEQ ID NO.2:SEQ ID NO.1：5-GTGATTTAAGAGGACT-3SEQ ID NO.2：5-TGGAGAGGGTGCGAAC-3。
- The test kit of 2.APRIL gene real-time PCR detection, is characterized in that: test kit comprises and a kind of detects solution, and it detects solution and contains SYBR Premix Ex Taq reagent, specification is 2 ×; Forward primer 10 μMs; Reverse primer 10 μMs; Rox Dye reagent, specification is 50 ×; Above-mentioned positive anti-primer as claimed in claim 1.
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| CN201310493575.7A CN103484559B (en) | 2013-10-17 | 2013-10-17 | Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene |
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| CN201310493575.7A CN103484559B (en) | 2013-10-17 | 2013-10-17 | Primer and kit for real-time fluorescence PCR (polymerase chain reaction) detection of APRIL (a proliferation inducing ligand) gene |
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