CN102965448A - Diabetes pathologic evolution early-stage SFRP4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization assay kit, as well as assay method and application thereof - Google Patents
Diabetes pathologic evolution early-stage SFRP4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization assay kit, as well as assay method and application thereof Download PDFInfo
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- CN102965448A CN102965448A CN2012105341516A CN201210534151A CN102965448A CN 102965448 A CN102965448 A CN 102965448A CN 2012105341516 A CN2012105341516 A CN 2012105341516A CN 201210534151 A CN201210534151 A CN 201210534151A CN 102965448 A CN102965448 A CN 102965448A
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Abstract
The invention discloses an in-situ hybridization assay kit comprising a hybridization probe and a marker. The invention further discloses a method for in-situ hybridization assay of SFRP4 gene mRNA (messenger ribonucleic acid) which is closely related to the early stage of pathologic evolution of diabetes by using the kit, and the method comprises the following steps of: (1) enabling RNA (ribonucleic acid) to be assayed in a substrate to be in contact with the hybridization probe under the condition that the hybridization probe and a target sequence can form a stable hybridcomplex so as to form the hybridcomplex; and (2) assaying the hybridcomplex. According to the kit and the assay method, disclosed by the invention, the expression level of an SFRP4 gene can be assayed on the mRNA level, the assay can be earlier than existing clinical biochemical assay indexes, the diabetes pathologic evolution early-stage mRNA level screening can be truly realized, the purposes of preventive diagnosis and treatment of the diabetes can be further achieved, and the diabetes can be nipped in the bud. Simultaneously, the assay method disclosed by the invention is simple, convenient, low in cost and convenient to popularize and apply in hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to change with diabetes Pathologic mrna expression the correlation detection technology of (Pathologic process).
Background technology
The latest data of statistics in 2011 shows that the diabetes that has made a definite diagnosis in the whole nation have 9,240 ten thousand, and the crowd of pre-diabetes has 1.48 hundred million.Diabetic complication is a kind of common chronic complicating diseases, to be changed by the diabetes pathology, consequence is quite serious, being the modal complication of diabetes such as pedopathy (foot gangrene, amputation), ephrosis (renal failure, uremia), illness in eye (smudgy, blind), encephalopathic (cerebrovascular disease), heart trouble, tetter, venereal disease etc., is the principal element that causes diabetic subject's death.The pathophysiological change of diabetes is owing to hypoinsulinism and organizes sugar to utilize obstacle, causes the too high metabolic disturbance of blood sugar.Diabetes have two kinds of primary and Secondary cases.The primary cause of disease is not yet clear; May be relevant with the factor such as heredity, Secondary cases sees chronic pancreatitis, cancer, hemochromatosis, the most of excision of pancreas, Anterior pituitary superfunction, adrenocorticotropin superfunction, Adrenal Pheochromocytoma, alpha Cell of islet knurl, gestational diabetes etc.Recently scientists is all furtherd investigate the pathogenetic gene physiopathology level that is placed on of diabetes, some more relevant genes with onset diabetes have been found, recent discovery such as the SFRP4 gene, SFRP4 is the molecule that works in the body inflammatory process, this is that people connect itself and the ill risk of type-II diabetes first, and the SFRP4 gene belongs to coding secreted frizzled related protein 4.The expression of SFRP4 and marker of inflammation, its pancreas islet discharges interleukin-1 ' beta ' to stimulate.Whole body SFRP4 raises and to cause impaired glucose tolerance, by reduce the Ca(2 that islet cells expresses+) passage, and suppress the Regular Insulin exocytosis, thereby SFRP4 provides the contact between impaired of pancreas islet inflammation and insulin secretion.In addition, the protein that increases in the serum is diagnosed several years ago from T2D patient, and this shows that SFRP4 T2D islet function obstacle may be a potential biomarker.The researchist compares diabetic subject and ND's beta cell (being responsible for producing the cell of Regular Insulin), find that the level of SFRP4 albumen in diabetic subject's body is obviously higher, find the people that the SFRP4 protein level is higher than mean value in the blood, suffer from the possibility of diabetes in the coming years than the below average people Gao Wubei of SFRP4.This also is to prove that first the inflammation in the beta cell is relevant with diabetes, and the researchist points out that chronic low grade inflammation has weakened the beta cell, makes the Regular Insulin that they no longer can the production capacity.Undoubtedly, have many factors to participate in this weakening process, and SFRP4 albumen is exactly one of them.The researchist detected the SFRP4 protein level in above-mentioned ND's blood every 3 years, and such detection has been carried out three times altogether.Studies show that SFRP4 is higher than among the people of mean level (ML) in the blood has 37% to suffer from diabetes during studying.And in those blood among the below average people of SFRP4, only have 9% during studying, to suffer from diabetes.Show that SFRP4 albumen can be used as the strong risk indicator of diabetes, is expected to Diagnostic Time is shifted to an earlier date the several years.The researchist has also resolved the mechanism of SFRP4 infringement insulin secretion, points out that the rising of SFRP4 level can reduce calcium channel in the expression of pancreas islet and suppress insulin secretion, causes glucose tolerance to lower.This albumen sign can not only be indicated the ill risk of diabetes, can also reflect the process of disease.Studies show that this index is independent of other known type-II diabetes risks and assumptions, for example obesity and age etc.The researchist thinks that this index can pass through simple blood testing, and the middle-aged people of indication normal type suffers from the risk of diabetes.This will be to their the stronger power mode of making the life better as early as possible, with prevent diabetes.In addition, this research also helps people to develop the novel method for the treatment of type-II diabetes, thereby namely blocks SFRP4 albumen in the beta cell to reduce the inflammation Cell protection.Clinically when the patient is diagnosed as type-II diabetes, often this disease has developed in patient body for many years already, and blood vessel and eye having been caused injury, the early stage risk that early examination goes out diabetes is of great value, like this people's Sex therapy that just can employ prevention as early as possible.
The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes the major disease mortality ratio not fallen is to accomplish real early stage diagnosis and treatment.Therefore, the horizontal kit for screening of genes involved of developing for onset diabetes early stage has very large clinical value.
Along with molecular biotechnology is day by day perfect, functional genomics, the deep expansion of the research such as disease genomics in order to seek more early stage diagnosing diabetes, treatment diabetes and prevent diabetes, makes great progress.So far, we might do more accurate early screening and diagnosis in the one-level functional transcription product mRNA of gene level, give a forecast and examination at pre-diabetes, so just the sickness rate of diabetes can be lowered, can greatly reduce social cost, further improve national health-physical fitness.
The contriver finds to use Double Labelling Technique under study for action, synchronous detection protein with detect mRNA, sometimes mRNA has expression, but protein sometimes do not transcribe out, they sometimes exist expresses the space-time different phenomenon, it is more accurate just to detect mRNA.
The inventor finds that under study for action the SFRP4 gene has obvious expression amount to change diabetic subject, high risk population, normal control people, has very important clinical meaning with SFRP4 in earlier stage as examination diabetes pathology.
The contriver is in long-term research, drawn a kind of new concept, the clinical diagnosis and treatment pattern of major disease must change, can not only stop present treating the disease affected (diagnosis and treatment after the morbidity), accomplish preventative diagnosis and treatment, accomplish treating the disease affected, only in this way could reduce the M ﹠ M of major disease, reduce social cost and medical treatment cost.Therefore, the contriver innovates theoretical and technical in the horizontal kit for screening of the mRNA of development and production major disease and medicine.Particularly screen clinical samples (normal population, high risk population, Disease), broken through healthy tissues and pathological tissue consistency research and development thinking relatively, seek and develop the pathology mRNA level in early stage, develop closely related with disease early gene physiopathology, and the extremely important mRNA target of clinical meaning, disease is clinically formed the preventative diagnosis and treatment that rear diagnosis and treatment pattern becomes major disease, striven for time and the space of diagnosis of disease, reach preventing disease.
The detection technique and the test kit that adopt hybridization in situ technique and groupization immunization method to detect SFRP4 gene mRNA horizontal expression amount according to existing documents and materials have no report.
The inventor is in the requirement for the novelty invention, designed (diabetic subject, high risk population, normal control) different pieces of information example group, detect with hybridization in situ technique, the result shows above diabetic high expression level, the high risk population has and expresses in various degree 10-20%, and normal control all is not express.Show that the SFRP4 gene is the important symbol thing of diabetes pathology examination in early stage.
Hybridization in situ technique (in situ hybridization) is that molecular biology and Cytochemical Technique are combined, take the nucleic acid molecule of mark as probe, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark, under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is the molecule of known array or sequence the unknown but known nucleic acid molecule (though indefinite this molecule full sequence of molecule, but known its for what target molecule), the kind of probe can be divided into again dna probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe by the properties of nucleic acids difference.For the ease of spike, probe must with certain means mark in addition, be beneficial to later detection.Marker commonly used comprises radionuclide and non-radioactive marker's two large classes.Isotopic label commonly used has
3H,
35S,
125I and
32P.Although isotopic label has the advantages such as susceptibility is high, back end is comparatively clear, because radio isotope all can damage human and environment, the trend that is replaced by heterotope is arranged recently.At present the most frequently used in the heterotope marker have three kinds of vitamin H, digoxin and fluoresceins.The method that detects these markers all is extremely sensitive.
Can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization according to used probe and the difference that will detect nucleic acid.No matter but the hybridization of any form, all must be through five large processes, namely histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the Crossing system of RNA-RNA, and synthetic probe (RNA) and the target RNA that detects are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research with observe, start and termination place the result not impact of residue on detecting.
In view of at present clinically human major disease diagnosis major part be late period, treatment also is late period, the diagnosis and treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the clinically diagnosis and treatment pattern of major disease, become preventative preventiveing treatment of disease from treating the disease affected, reach the preventative diagnosis and treatment purpose of major disease, made the breakthrough of novelty in theory and technology, provide disease to change the horizontal examination technology of mRNA, making has had a new disease to change the technology of the horizontal examination of mRNA in early stage clinically, for the diagnosis and treatment of clinical major disease are raced against time and the space, realizes great prevention.
In sum, purpose of the present invention at first provides a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and marker.Secondly, the present invention also will provide the mentioned reagent box to be used for the in situ hybridization detection method relevant with pre-diabetes examination and early stage early warning.
Summary of the invention
For realizing purpose of the present invention, technical scheme of the present invention is as follows:
The present invention at first provides a kind of hybridization in situ detection kit, it comprises hybridization probe and marker, wherein, described hybridization probe sequence is sequence shown in the sequence table SEQ ID NO.1, it is one section sequence among the SFRP4 gene C DS, 420bp, SFRP4 gene are sequence shown in the sequence table SEQ ID NO.2, are positioned at karyomit(e) 7p14.1 " on.
A preferred version of test kit of the present invention is that described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Diabetes pathology SFRP4 kit for screening in early stage using value of the present invention is that pre-diabetes examination and early warning are had extremely important clinical meaning, further cooperates the clinical prophylactic treatment of doing.
The present invention also provides a kind of detection method of SFRP4 gene hybridization in situ, may further comprise the steps:
(1) described hybridization probe and target sequence can form under the condition of stablizing hybridization complex in the above, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
Detection method of the present invention, wherein preferably, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood cell sample or other organ-tissue cell specimen.More preferably be that described blood preparation or other organ-tissue cell specimen are from diabetic subject, high risk population, healthy normal population.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, take SFRP4 as detected object, synthesising probing needle is one section sequence among the SFRP4 gene C DS, and 429bp, the substrate of detection are the expression amounts of blood of human body sample white corpuscle or histiocytic RNA.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of SFRP4.According to the expression amount of the above RNA of immunohistochemical methods colour developing judgement after the hybridization, normal people SFRP4 gene is not expressed, namely without colour developing, express at the diabetic height, and a large amount of dyeing, the high risk population has slight dyeing.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, the compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and concrete operation step is to carry out quantitative analysis, report the test under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, automatically adds Digestive system;
3). instrument discards liquid automatically, and is automatically rear fixing;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, automatically cleans;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, automatically cleans;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, automatically cleans colour developing;
10). take out the mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with SFRP4 synthetic nucleic acid probe digoxigenin labeled (cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized Endogenous Biotin to do the shortcomings such as sorrow in the crossover process in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, method with immunohistochemical methods develops the color again, under light microscopic, observe existence and the location of RNA, according to the cell count of dyeing, judge the expression amount of purpose RNA.
The inventive method is nucleic acid hybridization in situ technology commonly used at present, and the method is used for determining the diabetes Pathologic mRNA variable quantity in early stage by the SFRP4 expression amount in the detection substrate cell, and early warning diabetes gene physiopathology changes.Because SFRP4 does not express in the normal people, at diabetic subject's high expression level, if having, the high risk population shows the risk that occurrence of diabetes is arranged, in time carry out (intervention) prophylactic treatment.
A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect:
Clinical meaning of the present invention is that more early stage tracking detects the pathogenetic risk of glycosuria.Before biochemical indicator does not produce unusually, accomplish early the information acquisition that above gene mRNA expression is unusual, give real early warning of clinician and prophylactic treatment reference and guidance.
In addition, characteristics highly sensitive, high specificity that test kit provided by the invention has, simultaneously, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Description of drawings
Fig. 1 is SFRP4 gene hybridization in situ techniqueflow chart of the present invention.
Fig. 2 is that diabetic SFRP4 expresses picture in the embodiment of the invention.
Fig. 3 is high risk population's picture of embodiment of the invention mild or moderate blood sugar increasing.
Fig. 4 is that normal people SFRP4 expresses picture in the embodiment of the invention.
Embodiment
Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that the following examples are used for explanation and non-limiting content of the present invention, any pro forma change or accommodation will fall into protection scope of the present invention.
Embodiment 1
Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises with the hybridization probe of SFRP4 gene design, marker, specification sheets, wherein:
The probe mark thing of present embodiment is selected digoxin.
The test kit hybridization solution forms:
Digestive system | 100 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
Protection liquid | 100 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
Prehybridization solution | 1300 μ L/ pipe | 2 pipe/boxes | Colourless transparent liquid |
The justice hybridization solution | 10 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
The antisense hybridization solution | 10 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
Confining liquid | 1000 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
The alkaline phosphatase enzyme antibody | 1 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
Developer A | 175 μ L/ pipe | 1 pipe/box | Yellow liquid |
Developer B | 320 μ L/ pipe | 1 pipe/box | Colourless transparent liquid |
The damping fluid I | The 90mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
The damping fluid II | The 80mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
The damping fluid III | The 20mL/ bottle | 3 bottle/boxes | Light yellow or colourless transparent liquid |
The damping fluid IV | The 90mL/ bottle | 1 bottle/box | Light yellow or colourless transparent liquid |
Stationary liquid | The 90mL/ bottle | 1 bottle/box | Colourless transparent liquid |
The positive control sample | 6/box |
The reagent preparation working concentration
1). 10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;
2). 20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;
Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;
3). 10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;
4) .10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * the damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).
Embodiment 2
Use the nucleic acid hybridization in situ detection method each organized the implementation process of blood preparation SFRP4 gene expression amount:
1). get two of samples to be measured;
2). add Digestive system (Digestive system 100 μ L add 1 * damping fluid I 99.9ml, are working concentration) 50 ml in glass jar, 37 ℃ of water-bath preheating 10min put 16 slides into, process 12 min, use 1 * damping fluid I to wash 5min again for 37 ℃;
3). (protection liquid 1ml adds 1 * damping fluid to the protection liquid with 0.2%
, 99ml is working concentration) and wash 10min, tri-distilled water is washed the above process of 5min(and is all carried out at glass jar), take out slide, allow its seasoning;
4). slide is put into moisture preservation box, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered covers tightly moisture preservation box, is placed in 42 ℃ of constant water bath box more than the 3h;
5). take out slide, discard cover glass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered covers tightly moisture preservation box, is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
In 42 ℃ of constant water bath box, wash twice with 2 * damping fluid II, each 15min;
In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II;
In 42 ℃ of constant water bath box, wash twice with 0.1 * damping fluid II, each 15min;
8). wash 30s with 1 * damping fluid III, take out slide, seasoning;
9). slide is put into moisture preservation box, add 0.5% confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III), 100 μ L/ sheets, cover tightly moisture preservation box, at room temperature act on 30min.(this step need not add cover glass);
10). take out slide, wash 30s with 1 * damping fluid III, seasoning;
11). slide is put into moisture preservation box, add X-AP antibody and (get a pipe alkaline phosphatase enzyme antibody, to wherein adding 1.8ml 1 * damping fluid III) 100 μ L/ sheets, cover tightly moisture preservation box and at room temperature act on 30min, time can not be long, otherwise can produce false positive (this step need not add cover glass);
12). take out slide, wash 3 times with 1 * damping fluid III, each 15min;
13). wash 2min with 1 * damping fluid IV, add developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL 1 * damping fluid IV, mixing), room temperature lucifuge 16h is to more than the 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is with the goal gene digoxigenin labeled, become the RNA nucleic acid probe, the RNA nucleic acid to be measured of probe and human leukocytes is hybridized, method with immunohistochemical methods develops the color again, therefore under light microscopic, observe existence and the location of RNA, according to the cell count of dyeing, judge the expression amount of purpose RNA.
20 of diabetics, 20 of high risk population (slight blood sugar increasing people), 20 of Normal groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result represents that all diabetic subject SFRP4 gene mRNA expression amounts are high, and cell dyeing is dark; The high risk population expresses slightly and reduces, decimal dyeing; Normal group SFRP4 gene is not expressed, and cell does not dye, and concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
The diabetes number | Expression amount % | High-risk number | Expression amount % | Normal number | Expression amount % |
1 | 82 | 1 | 8 | 1 | 0 |
2 | 62 | 2 | 20 | 2 | 0 |
3 | 80 | 3 | 22 | 3 | 0 |
4 | 67 | 4 | 8 | 4 | 0 |
5 | 86 | 5 | 13 | 5 | 0 |
6 | 65 | 6 | 9 | 6 | 0 |
7 | 70 | 7 | 16 | 7 | 0 |
8 | 77 | 8 | 13 | 8 | 0 |
9 | 78 | 9 | 17 | 9 | 0 |
10 | 63 | 10 | 18 | 10 | 0 |
11 | 60 | 11 | 14 | 11 | 0 |
12 | 64 | 12 | 13 | 12 | 0 |
13 | 74 | 13 | 12 | 13 | 0 |
14 | 77 | 14 | 24 | 14 | 0 |
15 | 66 | 15 | 15 | 15 | 0 |
16 | 70 | 16 | 11 | 16 | 0 |
17 | 76 | 17 | 18 | 17 | 0 |
18 | 68 | 18 | 16 | 18 | 0 |
19 | 78 | 19 | 10 | 19 | 0 |
20 | 60 | 20 | 8 | 20 | 0 |
。
SEQUENCE LISTING
<110〉Rui bends biotechnology (Shanghai) Co., Ltd.
<120〉the horizontal hybridization in situ detection kit of mRNA and the detection method of diabetes Pathologic SFRP4 gene in early stage
And application
<130> 。
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 420
<212> DNA
<213> Homo sapiens
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tgctgcgctt cttcctctgt gccatgtacg cgcccatttg caccctggag ttcctgcacg 180
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ggatagacat cacaccagac atgatggtac aggaaaggcc tcttgatgtt gactgtaaac 420
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cttgtcaaca ccctcttaag cagcaccaga aacagtgagt ttgtctgtac cattaggagt 2220
taggtactaa ttagttggct aatgctcaag tattttatac ccacaagaga ggtatgtcac 2280
tcatcttact tcccaggaca tccaccctga gaataatttg acaagcttaa aaatggcctt 2340
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cttttgctaa cacagtaagc atgtattctc tataaggcat tcaataaatg cacaacgccc 2640
aaaggaaata aaatcctatc taatcctact ctccactaca cagaggtaat cactattagt 2700
attttggcat attattctcc aggtgtttct tatgcactta taaaatgatt tgaacaaata 2760
aaactaggaa cctgctatac atgtgtttca taacctgcct cctttgcttg gccctttatt 2820
gagataagtt ttcctgtcaa gaaagcagaa accatctcat ttctaacagc tgtgttatat 2880
tccatagtat gcattactca acaaactgtt gtgctattgg atacttaggt ggtttcttca 2940
ctgacaatac tgaataaaca tctcaatagt caaa 2974
Claims (10)
1. a hybridization in situ detection kit comprises hybridization probe and marker, it is characterized in that, described hybridization probe is sequence shown in the sequence table SEQ ID NO.1.
2. test kit as claimed in claim 1 is characterized in that, described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
3. test kit as claimed in claim 1 is characterized in that, this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1 is characterized in that, this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1 is characterized in that, this test kit also comprises developer.
6. SFRP4 gene hybridization in situ detection method is characterized in that the method may further comprise the steps:
(1) can form under the condition of stablizing hybridization complex at hybridization probe claimed in claim 1 and target sequence, RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
7. detection method as claimed in claim 6 is characterized in that, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6 is characterized in that, described substrate is selected people's blood cell sample.
9. detection method as claimed in claim 6 is characterized in that, described blood cell sample is selected from diabetic subject, high risk population, normal people's sample.
10.SFRP4 the application of gene in preparation detection diabetes in situ hybridization test kit.
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CN2012105341516A CN102965448A (en) | 2012-12-12 | 2012-12-12 | Diabetes pathologic evolution early-stage SFRP4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization assay kit, as well as assay method and application thereof |
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CN2012105341516A CN102965448A (en) | 2012-12-12 | 2012-12-12 | Diabetes pathologic evolution early-stage SFRP4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization assay kit, as well as assay method and application thereof |
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CN2012105341516A Pending CN102965448A (en) | 2012-12-12 | 2012-12-12 | Diabetes pathologic evolution early-stage SFRP4 gene mRNA (messenger ribonucleic acid) level in-situ hybridization assay kit, as well as assay method and application thereof |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101370826A (en) * | 2006-02-09 | 2009-02-18 | 诺瓦提斯公司 | Secreted frizzled related protein-4 (SFRP-4) protein binding agents |
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2012
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101370826A (en) * | 2006-02-09 | 2009-02-18 | 诺瓦提斯公司 | Secreted frizzled related protein-4 (SFRP-4) protein binding agents |
Non-Patent Citations (3)
Title |
---|
J. M. DRAKE, ET AL.: "The role of sFRP4, a secreted frizzled-related protein, in ovulation", 《APOPTOSIS》 * |
M FUJITA, ET AL.: "Differential expression of secreted frizzled-related protein 4 in decidual cells during pregnancy", 《JOURNAL OF MOLECULAR ENDOCRINOLOGY》 * |
TAMAN MAHDI, ET AL.: "Secreted Frizzled-Related Protein 4 Reduces Insulin Secretion and Is Overexpressed in Type 2 Diabetes", 《CELL METABOLISM》 * |
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