CN102943124A - In-situ hybridization detection kit and method for SORL-1 gene mRNA level in early-stage pathologic evolution of Diabetes mellitus and application of kit and method - Google Patents

Abstract

The invention discloses an in-situ hybridization detection kit which comprises a hybridization probe and a marker. The invention also discloses a method used for carrying out in-situ hybridization detection on SORL-1 gene mRNA which is closely related to the early-stage pathologic evolution of diabetes mellitus by using the kit. The method comprises the following steps of: (1) under the condition that a stable hybrid complex is formed by the hybridization probe and a target sequence, enabling RNA to be tested in a substrate to contact the hybridization probe to form a hybrid complex; and (2) detecting the obtained hybrid complex. The kit and the detection method can be used for detecting the expression quantity of SORL-1 gene at the mRNA level and can detect more early stage than the existing clinical biochemical detection indexes and realize mRNA level screening in early-stage pathologic evolution of diabetes mellitus indeed, thus achieving the aim of preventive treatment of diabetes mellitus and eliminating diabetes mellitus in the bud. Furthermore, the detection method provided by the invention is simple, convenient and low in cost, thus being favorably popularized and applied in hospital.

Description

The horizontal hybridization in situ detection kit of mRNA and detection method and the application of diabetes Pathologic SORL-1 gene in early stage

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, as the SORL-1 gene be recently find relevant with the trouble diabetes, find that the level of SORL-1 albumen in diabetic subject's body obviously increases, 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.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, detecting mRNA can be more accurate.

The inventor finds that under study for action the SORL-1 gene has obvious expression amount to change diabetic subject, high risk population, normal control people, has very important clinical meaning with SORL-1 in earlier stage as examination diabetes pathology.SORL-1 is high expression level in diabetic subject's pathology.He does in the diabetes early warning very important clinical meaning.

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 SORL-1 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 SORL-1 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, is a section among the SORL-1 gene order CDS, from 601-1080bp, be total to 480bp, the SORL-1 gene is sequence shown in the sequence table SEQ ID NO.2, is positioned at karyomit(e) 11q23.2-24.2 " on, total length 10973bp.

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 SORL-1 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 SORL-1 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 SORL-1 as detected object, synthesising probing needle is sequence shown in the sequence table SEQ ID NO.1, and 480bp, 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 SORL-1.According to the expression amount of the above RNA of immunohistochemical methods colour developing judgement after the hybridization, normal people SORL-1 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 SORL-1 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 SORL-1 expression amount in the detection substrate cell, and early warning diabetes gene physiopathology changes.Because SORL-1 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 SORL-1 gene hybridization in situ techniqueflow chart of the present invention.

Fig. 2 is that diabetic SORL-1 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 SORL-1 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 SORL-1 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 SORL-1 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 SORL-1 gene mRNA expression amounts are high, and cell dyeing is dark; The high risk population expresses slightly and reduces, decimal dyeing; Normal group SORL-1 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	70	1	62	1	0
2	60	2	14	2	0
						3	64	3	20	3	0
4	60	4	15	4	0
						5	62	5	19	5	0
6	68	6	16	6	0
						7	65	7	18	7	0
8	56	8	14	8	0
						9	68	9	18	9	0
10	68	10	14	10	0
						11	64	11	16	11	0
12	69	12	14	12	0
						13	62	13	20	13	0
14	68	14	22	14	0
						15	60	15	19	15	0
16	56	16	14	16	0
						17	70	17	16	17	0
18	70	18	20	18	0
						19	62	19	12	19	0
20	70	20	16	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 SORL-1 gene in early stage

And application

<130> 。

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 480

<212> DNA

<213> Homo sapiens

<400> 1

aataggagtg aagctgttat cgcccagttc taccacagcc ctgcggacaa caagcggtac 60

atctttgcag acgcttatgc ccagtacctc tggatcacgt ttgacttctg caacactctt 120

caaggctttt ccatcccatt tcgggcagct gatctcctcc tacacagtaa ggcctccaac 180

cttctcttgg gctttgacag gtcccacccc aacaagcagc tgtggaagtc agatgacttt 240

ggccagacct ggatcatgat tcaggaacat gtcaagtcct tttcttgggg aattgatccc 300

tatgacaaac caaataccat ctacattgaa cgacatgaac cctctggcta ctccactgtc 360

ttccgaagta cagatttctt ccagtcccgg gaaaaccagg aagtgatcct tgaggaagtg 420

agagattttc agcttcggga caagtacatg tttgctacaa aggtggtgca tctcttgggc 480

<210> 2

<211> 10973

<212> DNA

<213> Homo sapiens

<400> 2

cccggagcgg cgcgggcggc ctggagcccc gggagcggcg cgcgcggtcc cggcccagcg 60

gctctcctgg cctcgcgctg cacattctct cctggcggcg gcgccacctg cagtagcgtt 120

cgcccgaaca tggcgacacg gagcagcagg agggagtcgc gactcccgtt cctattcacc 180

ctggtcgcac tgctgccgcc cggagctctc tgcgaagtct ggacgcagag gctgcacggc 240

ggcagcgcgc ccttgcccca ggaccggggc ttcctcgtgg tgcagggcga cccgcgcgag 300

ctgcggctgt gggcgcgcgg ggatgccagg ggggcgagcc gcgcggacga gaagccgctc 360

cggaggaaac ggagcgctgc cctgcagccc gagcccatca aggtgtacgg acaggttagt 420

ctgaatgatt cccacaatca gatggtggtg cactgggctg gagagaaaag caacgtgatc 480

gtggccttgg cccgagatag cctggcattg gcgaggccca agagcagtga tgtgtacgtg 540

tcttacgact atggaaaatc attcaagaaa atttcagaca agttaaactt tggcttggga 600

aataggagtg aagctgttat cgcccagttc taccacagcc ctgcggacaa caagcggtac 660

atctttgcag acgcttatgc ccagtacctc tggatcacgt ttgacttctg caacactctt 720

caaggctttt ccatcccatt tcgggcagct gatctcctcc tacacagtaa ggcctccaac 780

cttctcttgg gctttgacag gtcccacccc aacaagcagc tgtggaagtc agatgacttt 840

ggccagacct ggatcatgat tcaggaacat gtcaagtcct tttcttgggg aattgatccc 900

tatgacaaac caaataccat ctacattgaa cgacatgaac cctctggcta ctccactgtc 960

ttccgaagta cagatttctt ccagtcccgg gaaaaccagg aagtgatcct tgaggaagtg 1020

agagattttc agcttcggga caagtacatg tttgctacaa aggtggtgca tctcttgggc 1080

agtgaacagc agtcttctgt ccagctctgg gtctcctttg gccggaagcc catgagagca 1140

gcccagtttg tcacaagaca tcctattaat gaatattaca tcgcagatgc ctccgaggac 1200

caggtgtttg tgtgtgtcag ccacagtaac aaccgcacca atttatacat ctcagaggca 1260

gaggggctga agttctccct gtccttggag aacgtgctct attacagccc aggaggggcc 1320

ggcagtgaca ccttggtgag gtattttgca aatgaaccat ttgctgactt ccaccgagtg 1380

gaaggattgc aaggagtcta cattgctact ctgattaatg gttctatgaa tgaggagaac 1440

atgagatcgg tcatcacctt tgacaaaggg ggaacctggg agtttcttca ggctccagcc 1500

ttcacgggat atggagagaa aatcaattgt gagctttccc agggctgttc ccttcatctg 1560

gctcagcgcc tcagtcagct cctcaacctc cagctccgga gaatgcccat cctgtccaag 1620

gagtcggctc caggcctcat catcgccact ggctcagtgg gaaagaactt ggctagcaag 1680

acaaacgtgt acatctctag cagtgctgga gccaggtggc gagaggcact tcctggacct 1740

cactactaca catggggaga ccacggcgga atcatcacgg ccattgccca gggcatggaa 1800

accaacgagc taaaatacag taccaatgaa ggggagacct ggaaaacatt catcttctct 1860

gagaagccag tgtttgtgta tggcctcctc acagaacctg gggagaagag cactgtcttc 1920

accatctttg gctcgaacaa agagaatgtc cacagctggc tgatcctcca ggtcaatgcc 1980

acggatgcct tgggagttcc ctgcacagag aatgactaca agctgtggtc accatctgat 2040

gagcggggga atgagtgttt gctgggacac aagactgttt tcaaacggcg gaccccccat 2100

gccacatgct tcaatggaga ggactttgac aggccggtgg tcgtgtccaa ctgctcctgc 2160

acccgggagg actatgagtg tgacttcggt ttcaagatga gtgaagattt gtcattagag 2220

gtttgtgttc cagatccgga attttctgga aagtcatact cccctcctgt gccttgccct 2280

gtgggttcta cttacaggag aacgagaggc taccggaaga tttctgggga cacttgtagc 2340

ggaggagatg ttgaagcgcg actggaagga gagctggtcc cctgtcccct ggcagaagag 2400

aacgagttca ttctgtatgc tgtgaggaaa tccatctacc gctatgacct ggcctcggga 2460

gccaccgagc agttgcctct caccgggcta cgggcagcag tggccctgga ctttgactat 2520

gagcacaact gtttgtattg gtccgacctg gccttggacg tcatccagcg cctctgtttg 2580

aatggaagca cagggcaaga ggtgatcatc aattctggcc tggagacagt agaagctttg 2640

gcttttgaac ccctcagcca gctgctttac tgggtagatg caggcttcaa aaagattgag 2700

gtagctaatc cagatggcga cttccgactc acaatcgtca attcctctgt gcttgatcgt 2760

cccagggctc tggtcctcgt gccccaagag ggggtgatgt tctggacaga ctggggagac 2820

ctgaagcctg ggatttatcg gagcaatatg gatggttctg ctgcctatca cctggtgtct 2880

gaggatgtga agtggcccaa tggcatctct gtggacgacc agtggattta ctggacggat 2940

gcctacctgg agtgcataga gcggatcacg ttcagtggcc agcagcgctc tgtcattctg 3000

gacaacctcc cgcaccccta tgccattgct gtctttaaga atgaaatcta ctgggatgac 3060

tggtcacagc tcagcatatt ccgagcttcc aaatacagtg ggtcccagat ggagattctg 3120

gcaaaccagc tcacggggct catggacatg aagattttct acaaggggaa gaacactgga 3180

agcaatgcct gtgtgcccag gccatgcagc ctgctgtgcc tgcccaaggc caacaacagt 3240

agaagctgca ggtgtccaga ggatgtgtcc agcagtgtgc ttccatcagg ggacctgatg 3300

tgtgactgcc ctcagggcta tcagctcaag aacaatacct gtgtcaaaga agagaacacc 3360

tgtcttcgca accagtatcg ctgcagcaac gggaactgta tcaacagcat ttggtggtgt 3420

gactttgaca acgactgtgg agacatgagc gatgagagaa actgccctac caccatctgt 3480

gacctggaca cccagtttcg ttgccaggag tctgggactt gtatcccact gtcctataaa 3540

tgtgaccttg aggatgactg tggagacaac agtgatgaaa gtcattgtga aatgcaccag 3600

tgccggagtg acgagtacaa ctgcagttcc ggcatgtgca tccgctcctc ctgggtatgt 3660

gacggggaca acgactgcag ggactggtct gatgaagcca actgtaccgc catctatcac 3720

acctgtgagg cctccaactt ccagtgccga aacgggcact gcatccccca gcggtgggcg 3780

tgtgacgggg atacggactg ccaggatggt tccgatgagg atccagtcaa ctgtgagaag 3840

aagtgcaatg gattccgctg cccaaacggc acttgcatcc catccagcaa acattgtgat 3900

ggtctgcgtg attgctctga tggctccgat gaacagcact gcgagcccct ctgtacgcac 3960

ttcatggact ttgtgtgtaa gaaccgccag cagtgcctgt tccactccat ggtctgtgac 4020

ggaatcatcc agtgccgcga cgggtccgat gaggatgcgg cgtttgcagg atgctcccaa 4080

gatcctgagt tccacaaggt atgtgatgag ttcggtttcc agtgtcagaa tggagtgtgc 4140

atcagtttga tttggaagtg cgacgggatg gatgattgcg gcgattattc tgatgaagcc 4200

aactgcgaaa accccacaga agccccaaac tgctcccgct acttccagtt tcggtgtgag 4260

aatggccact gcatccccaa cagatggaaa tgtgacaggg agaacgactg tggggactgg 4320

tctgatgaga aggattgtgg agattcacat attcttccct tctcgactcc tgggccctcc 4380

acgtgtctgc ccaattacta ccgctgcagc agtgggacct gcgtgatgga cacctgggtg 4440

tgcgacgggt accgagattg tgcagatggc tctgacgagg aagcctgccc cttgcttgca 4500

aacgtcactg ctgcctccac tcccacccaa cttgggcgat gtgaccgatt tgagttcgaa 4560

tgccaccaac cgaagacgtg tattcccaac tggaagcgct gtgacggcca ccaagattgc 4620

caggatggcc gggacgaggc caattgcccc acacacagca ccttgacttg catgagcagg 4680

gagttccagt gcgaggacgg ggaggcctgc attgtgctct cggagcgctg cgacggcttc 4740

ctggactgct cggacgagag cgatgaaaag gcctgcagtg atgagttgac tgtgtacaaa 4800

gtacagaatc ttcagtggac agctgacttc tctggggatg tgactttgac ctggatgagg 4860

cccaaaaaaa tgccctctgc ttcttgtgta tataatgtct actacagggt ggttggagag 4920

agcatatgga agactctgga gacccacagc aataagacaa acactgtatt aaaagtcttg 4980

aaaccagata ccacgtatca ggttaaagta caggttcagt gtctcagcaa ggcacacaac 5040

accaatgact ttgtgaccct gaggacccca gagggattgc cagatgcccc tcgaaatctc 5100

cagctgtcac tccccaggga agcagaaggt gtgattgtag gccactgggc tcctcccatc 5160

cacacccatg gcctcatccg tgagtacatt gtagaataca gcaggagtgg ttccaagatg 5220

tgggcctccc agagggctgc tagtaacttt acagaaatca agaacttatt ggtcaacact 5280

ctatacaccg tcagagtggc tgcggtgact agtcgtggaa taggaaactg gagcgattct 5340

aaatccatta ccaccataaa aggaaaagtg atcccaccac cagatatcca cattgacagc 5400

tatggtgaaa attatctaag cttcaccctg accatggaga gtgatatcaa ggtgaatggc 5460

tatgtggtga accttttctg ggcatttgac acccacaagc aagagaggag aactttgaac 5520

ttccgaggaa gcatattgtc acacaaagtt ggcaatctga cagctcatac atcctatgag 5580

atttctgcct gggccaagac tgacttgggg gatagccctc tggcatttga gcatgttatg 5640

accagagggg ttcgcccacc tgcacctagc ctcaaggcca aagccatcaa ccagactgca 5700

gtggaatgta cctggaccgg cccccggaat gtggtttatg gtattttcta tgccacgtcc 5760

tttcttgacc tctatcgcaa cccgaagagc ttgactactt cactccacaa caagacggtc 5820

attgtcagta aggatgagca gtatttgttt ctggtccgtg tagtggtacc ctaccagggg 5880

ccatcctctg actacgttgt agtgaagatg atcccggaca gcaggcttcc accccgtcac 5940

ctgcatgtgg ttcatacggg caaaacctcc gtggtcatca agtgggaatc accgtatgac 6000

tctcctgacc aggacttgtt gtatgcaatt gcagtcaaag atctcataag aaagactgac 6060

aggagctaca aagtaaaatc ccgtaacagc actgtggaat acacccttaa caagttggag 6120

cctggcggga aataccacat cattgtccaa ctggggaaca tgagcaaaga ttccagcata 6180

aaaattacca cagtttcatt atcagcacct gatgccttaa aaatcataac agaaaatgat 6240

catgttcttc tgttttggaa aagcctggct ttaaaggaaa agcattttaa tgaaagcagg 6300

ggctatgaga tacacatgtt tgatagtgcc atgaatatca cagcttacct tgggaatact 6360

actgacaatt tctttaaaat ttccaacctg aagatgggtc ataattacac gttcaccgtc 6420

caagcaagat gcctttttgg caaccagatc tgtggggagc ctgccatcct gctgtacgat 6480

gagctggggt ctggtgcaga tgcatctgca acgcaggctg ccagatctac ggatgttgct 6540

gctgtggtgg tgcccatctt attcctgata ctgctgagcc tgggggtggg gtttgccatc 6600

ctgtacacga agcaccggag gctgcagagc agcttcaccg ccttcgccaa cagccactac 6660

agctccaggc tggggtccgc aatcttctcc tctggggatg acctggggga agatgatgaa 6720

gatgccccta tgataactgg attttcagat gacgtcccca tggtgatagc ctgaaagagc 6780

tttcctcact agaaaccaaa tggtgtaaat attttatttg ataaagatag ttgatggttt 6840

attttaaaag atgcactttg agttgcaata tgttattttt atatgggcca aaaacaaaaa 6900

acaaaaaaaa aaaaaaggaa agaaaggaat gaataaactt tgtagtaatc aactgtgaac 6960

ttcaaaccag gttgatttta gtaacccaat tgctttgatt tgacattaat gtagtcttac 7020

agggctgtgc ttgctgggca tgcttttacg tctgtgagat aatttcggtt cagtaaattg 7080

gccaatcttt ttatttttct aagacacaga aatgtattta ataaaaacct cgagagagtg 7140

atgggtggaa ccccttctcc ttgaaagtgt gtacagatat tccattttgt ttggatatag 7200

tttataggaa agtgtgtgga tgtattatgg cggaaggttt ctttatgtta ttttgttaat 7260

ttattgggac tctgtgtaag gccaggcttt agtggtcatt agacaccaca tgtgttatga 7320

gccccttacc catagggttg ggggtgggaa gagaagcata tttttttgcc attccggaag 7380

caatccattt ttattcactt gtgtgtcatg taatggtctt tggcaggaga gagcactgag 7440

tcattgctgg agttcagttc aacagagctg cagcttggga agccctgtaa gcccacagct 7500

tcctctctta tattaattga tggaatttta ctgtatgtgc ctctgtacaa gatgtagctt 7560

tgagagctac aaaatgataa cactgcttta ttacacactg gtttcattgt cattgcaaaa 7620

acttaccctg gttgtggggg agagttctag atctgtgcca tgatccatac actggctaat 7680

agagtacata atttttccat tttccatttt ttgtttttac ttactactga aggatctcag 7740

atgtaaaatt atgtatttgg tttgagatgg ccacttattg tccttaaaaa tccatactga 7800

tatatgcagt cattttgaat tggacagtgc cttctctttt tttttctcct cttcttccat 7860

ctccctcacc catgccccca cccaatctaa agagacagtg ctgtacattc tcatagagat 7920

agagaagatc taaaaagttg agactactca atccagttaa caacagcagg agcactagag 7980

tttgttcatt tattctctct gtaaaacaag ctgtgctttt tttcttctgc ctttaaaatg 8040

ccacccgtgt attcaaacca tggccacttg atacttatgt agaatccatc gtgggctgat 8100

gcaagccctt tatttaggct tagtgttgtg ggcaccaatg tcgagcatcg ttgtgacttg 8160

tgctgtatga ttctcactga agaatttcct ttcagccaag aagcagtgag gtctgggaat 8220

attccaaagt catgtctctg aatatgtgtc cttgacgtgc aagctttgta aaaccccatc 8280

cccgcttagg tgcgaggcat caccttctca caagtgttta gtttctttta accacaagta 8340

tcattcttgg gtgataatat agtttcattc tacttaggga ttgtttagaa aacaaagaaa 8400

gagccaatta aattttttag tttttgaaat ttttatttat atgtatactt agatgagtat 8460

tttaagctgt cgacctttag tttgccatac gggtaggact gtatttcatg ttaacaactg 8520

gtggtaatga taagccttct tctagcgtat tttctcttct ttcctgtcac tttcctaagt 8580

tttttttttt aaagactgga attttttttg gctttatctt gtcttaccgt agagatttgt 8640

tcaaaactct aagccctacc acctcccctt taataagctc tttaaatagt tgaatcatta 8700

acaacctggt gggaggcaag tcatttaatt gaaccactag gaagtgtatt ttcttttctt 8760

tttctgccaa ctttttggtg gcatttgtaa aagctgatat aaaaggctct gagatgttat 8820

tttcagttat tccataggca agccttttta cagagcatat gtctccagtt ggcagcttga 8880

gatatttccg agcatccggt tctagctacc agtgcctccc aatgcttagt gcacagtact 8940

gtagactggc catcacccct ctccttggaa aatgccactg tgctgtttga aaaaaagcag 9000

ccttttaggg ctagagtatt ttatataaac agaagagcta agttcctgaa gactaagcta 9060

gatagctgca gctatatgta aattgtatat ttttatgaac ttttgaagca cacactcctg 9120

tttccctctg tgtagctttg tggggatttc atgtatatat gctgtctgaa agaatccaga 9180

ggttggagtg ccaatagaaa atgaaaacaa atgccttgta ctacaggcag cctctgaagg 9240

tgaccacata actgtcttca ctgtgaccaa tcggagtccc tgcttgcttg tgaagaaggg 9300

gcttttgtac cttgttggag atgccacctc agaagttcac actgtgcagg aaaaaggttt 9360

tattctctcc tggcatacat tagaatgtca gatgcttgca tccatgtgga ccacgatggg 9420

cctctaaaaa ttggtgggca gggggtttgc ttatgagttt tctctggaaa ccgattttac 9480

tcctggatgt attgaatgcc ccttgagctt tatgagatac gagtccacat ggataaaatg 9540

ttagagagtg gagttctaca gaggattcca ggaagaggcc atgtctgtgc agtcctagtt 9600

ccagacaggt gagaagctcc aggaactact ggctaccttg acaagctggg taaatagtta 9660

tcattctggg taactggttg aaactctgac ttttggacaa gtaattcctg gggttctgtc 9720

tttggtagca tcaccaggga tatttgggtg ggacagacag aagacacaca gctgcctgtt 9780

ctctcctgcc catcatgttt ggcccactag atgaagctgt actcagcaat ttagggaatg 9840

taacccttct cagaactggc cattttcagg ggaagcttgg gagagcaata gtatggtgag 9900

ccccttagag atgagcgcct actccttctt ggcgaatgct gccttcagat gcttaccaag 9960

tggtcactgc atctagtaag attatatttc cagtacactt ccttagggca gaaacaccat 10020

cctatcaggt ttggtcagtc ccttcttcat gaagggagtc atggggaatt cctgaaaatt 10080

ttcttccttc tgcagacagt tggatgagtc ccttagagaa ggcatccaga gacataacta 10140

aactgaatat catcccatat tgattttagg aattgactct aaaactctgt gcagaatctt 10200

gtgttgggat tgtatcttga cattcctgtt gtgttatttt tcttaactgg agtgtgtgct 10260

gcctttcagg tacaattttt gtgtaataaa agccagtgca ttaagtttat atagactact 10320

ttctatgcaa gactgagata tggaatagat aggaagagat atgtactgct gggtacatgg 10380

acagtaagtg tgttttcaga tggagtacca gcaccgaaaa tgggttgagg gaggatgggt 10440

tgtatgtatg tttctgccca ctaattttga gcagccatat tatgaattaa atcgtcacag 10500

ccaagtaata acccaagaat ggtatgagtt tcatgtgtaa tagctcaaat ggaataagca 10560

tgaatgctgg agtggaccat tatcctcaaa tattctatgt cacttctcat ttaaagactc 10620

ttgttatgaa ctattagaaa ctttaggcaa aatcaaaagt atttgcggca aaataaaggc 10680

ctattctact cttatttaaa gtgaaacact gtatacttgt ttctctccaa agcgaaatta 10740

agtatttata atttcaattg cctcgataag tttccaagtc actgaaatct gctgaaggtt 10800

ttactgtatt gttgcacaac tttaagataa tttttgtctc aatgtcaact tttttcactg 10860

aataaaaatt taactgggtc aagaaaacac ctctttgaaa atccactgtc tctgtgtgtc 10920

tcgagctgtt ctttagagcg caataaagat ggctgacgca gtctccaaac ccc 10973

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. SORL-1 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.

Images