CN102943121A - In-situ hybridization detection kit and method for protein tyrosine phosphatase receptor type delta (PTPRD) 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 protein tyrosine phosphatase receptor type delta (PTPRD) 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 PTPRD gene at the mRNA level and can detect more early state 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, 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 PTPRD 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 genes more relevant to onset diabetes have been found, recently to find that to suffer from diabetes relevant with the Hans such as the PTPRD gene, find that the level of PTPRD 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 PTPRD gene has obvious expression amount to change diabetic subject, high risk population, normal control people, has very important clinical meaning with PTPRD in earlier stage as examination diabetes pathology.PTPRD 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 PTPRD 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 PTPRD 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.The advantages such as susceptibility is high although isotopic label has, back end is comparatively clear because radio isotope all can damage human and environment, have the trend that is replaced by heterotope 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 PTPRD gene order CDS, from 601-1080bp, be total to 480bp, the PTPRD gene is sequence shown in the sequence table SEQ ID NO.2, is positioned at chromosome 9p 23-p24.3 " on, total length 8257bp.

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 PTPRD 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 PTPRD 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 PTPRD as detected object, synthesising probing needle is the PTPRD gene order, 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 PTPRD.Judge the expression amount of above RNA according to immunohistochemical methods colour developing after the hybridization, normal people PTPRD 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 PTPRD 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 PTPRD expression amount in the detection substrate cell, and early warning diabetes gene physiopathology changes.Because PTPRD 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 to follow the tracks of more in early days to detect 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 PTPRD gene hybridization in situ techniqueflow chart of the present invention.

Fig. 2 is that diabetic PTPRD 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 PTPRD 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, and any pro forma change or accommodation will fall into protection scope of the present invention.

Embodiment 1

The in situ hybridization test kit for preparing the present embodiment according to ordinary method, this test kit comprise with the hybridization probe of PTPRD gene design, marker, specification sheets, wherein:

The probe mark thing of the 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 PTPRD 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, all diabetic subject PTPRD gene mRNA expression amounts are high, and cell dyeing is dark; The high risk population expresses slightly and reduces, decimal dyeing; Normal group PTPRD 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	75	1	17	1	0
2	60	2	18	2	0
						3	70	3	22	3	0
4	66	4	16	4	0
						5	76	5	18	5	0
6	64	6	12	6	0
						7	66	7	10	7	0
8	71	8	18	8	0
						9	76	9	20	9	0
10	66	10	19	10	0
						11	68	11	15	11	0
12	62	12	16	12	0
						13	76	13	23	13	0
14	70	14	20	14	0
						15	63	15	18	15	0
16	68	16	14	16	0
						17	74	17	16	17	0
18	62	18	18	18	0
						19	70	19	15	19	0
20	72	20	20	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 PTPRD 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

ggacacaagc aacaacaatg gtcgtattaa gcagttacga tcaggtggta caccaataag 60

aggagccctt cagattgagc agagtgaaga gtctgaccaa ggaaaatatg agtgtgttgc 120

caccaacagc gcgggcactc gctattccgc tcctgccaat ttatatgtca gagagctgcg 180

agaagttcgc cgtgtcccac caagattctc tatcccaccc actaatcatg aaatcatgcc 240

aggcggaagc gttaatatca cctgtgtggc cgtggggtca ccaatgcctt atgtaaagtg 300

gatgttgggg gcagaagatc tgacacctga agatgatatg ccaataggaa gaaatgtgct 360

agaactgaat gatgtaagac agtcagcaaa ttacacctgt gttgctatgt caacactggg 420

tgtcattgaa gcaatagcac agatcactgt caaagcctta cccaaacctc caggaactcc 480

<210> 2

<211> 8257

<212> DNA

<213> Homo sapiens

<400> 2

gctctgaaag tgttccagct gaaatttcag atcggacaga ctcgctgcgg ctccggaggc 60

agtgattcca agctgctcgc gcacgctgct gccaagctgc aggatggtgc acgtagccag 120

gctgctgctg ctgctcctca ctttcttcct ccgcacggat gctgagacac ctccaaggtt 180

tacacgaaca cccgttgatc agacaggggt ctctggcgga gttgcctctt tcatctgcca 240

agctacggga gacccaagac ctaaaattgt ctggaacaaa aaaggaaaga aagtcagcaa 300

tcagagattt gaggtaatag agtttgacga tgggtctgga tcagttctca gaatacaacc 360

cttacggact ccgagggatg aggccattta tgaatgtgtg gcctcaaata atgtgggaga 420

aataagtgta tccaccagac tcacagtttt gcgggaagat caaattccca ggggcttccc 480

taccattgac atgggcccac agttgaaggt ggttgagcgt actcgcacgg ccaccatgct 540

ttgtgcagcc agtggtaatc cggatccaga aatcacttgg tttaaagatt tcttacctgt 600

ggacacaagc aacaacaatg gtcgtattaa gcagttacga tcaggtggta caccaataag 660

aggagccctt cagattgagc agagtgaaga gtctgaccaa ggaaaatatg agtgtgttgc 720

caccaacagc gcgggcactc gctattccgc tcctgccaat ttatatgtca gagagctgcg 780

agaagttcgc cgtgtcccac caagattctc tatcccaccc actaatcatg aaatcatgcc 840

aggcggaagc gttaatatca cctgtgtggc cgtggggtca ccaatgcctt atgtaaagtg 900

gatgttgggg gcagaagatc tgacacctga agatgatatg ccaataggaa gaaatgtgct 960

agaactgaat gatgtaagac agtcagcaaa ttacacctgt gttgctatgt caacactggg 1020

tgtcattgaa gcaatagcac agatcactgt caaagcctta cccaaacctc caggaactcc 1080

tgtagtgacc gagagcacag ctacaagcat cacactgacg tgggactctg ggaaccctga 1140

gcctgtttct tattacataa ttcagcataa acctaaaaac tctgaggaac tttacaaaga 1200

aattgatggg gtggcgacca cacgctacag tgtcgctgga ctaagtccct actcggatta 1260

tgaattcagg gttgttgctg tcaataacat tgggcggggg cctcccagcg aacctgtgct 1320

aacacaaacc tcagagcaag caccatccag tgccccgagg gatgtccagg cacgaatgtt 1380

gagttcgacc accattttgg tacagtggaa ggaacctgaa gagccaaatg gacagatcca 1440

aggatataga gtttattata caatggatcc cactcaacat gtcaacaact ggatgaaaca 1500

caatgtagct gacagccaaa tcactactat tggcaactta gtgccccaga aaacatattc 1560

tgtcaaagtc ctggctttta cctcaattgg agatggtccc ctttcaagtg acatacaagt 1620

catcactcag acaggagtac cagggcagcc actaaacttc aaagcagaac ctgagtctga 1680

aacaagtatt ttgctctctt ggacacctcc acgttcagat accattgcca actatgaact 1740

ggtctacaaa gatggggagc atggagagga gcaacgaatt accattgagc cagggacatc 1800

atataggctg caaggactga aaccaaacag cttatactat ttccgtctgg ctgcacgctc 1860

ccctcaaggc ctgggtgctt ctactgcaga aatatcagct agaaccatgc agtcaatgtt 1920

tgcaaaaaat tttcatgtca aagcagtaat gaagacttcc gtgttgctgt cttgggagat 1980

tccagagaat tataactccg ccatgccttt caaaattctt tatgatgatg ggaaaatggt 2040

agaagaagtg gatggccgag ccacacagaa gttaattgtc aacctgaagc ctgagaaatc 2100

atattcattt gtgctgacaa atcgtggaaa cagtgctggt gggctgcagc acagggtcac 2160

ggcaaagact gcaccagatg tattacgtac caagcctgcc ttcattggga agaccaactt 2220

ggatggcatg attactgtgc aactgcctga agtacctgca aatgagaata taaaaggtta 2280

ctacataata attgtgcctt tgaagaaatc tcgcgggaaa tttatcaagc catgggagag 2340

tccagatgaa atggaattag atgagctgct taaggagata tctaggaagc gcagaagcat 2400

ccgttatggg agagaagttg aattaaagcc atatattgcc gctcactttg atgtccttcc 2460

cactgagttc accctggggg atgacaagca ttatggtgga tttacaaaca agcaactcca 2520

aagtggtcaa gaatatgtct tctttgtgtt agcagtaatg gaacatgcag agtctaagat 2580

gtatgcaacc agcccttact ccgaccccgt ggtgtcaatg gatctggatc cgcagccaat 2640

cacggatgaa gaagaaggct tgatctgggt tgtaggtcct gtccttgcag tggtctttat 2700

catctgcatt gtcattgcta ttcttcttta taaaagtaaa cccgacagga agagggcaga 2760

gtccgactct agaaaaagca gcataccgaa caataaggag atcccttcac accacccaac 2820

agaccctgta gaactgaggc gccttaactt tcaaacaccg ggtatggcta gccatcctcc 2880

aatacccatc ttggaacttg cagaccacat tgaaagattg aaagcaaatg acaacttgaa 2940

gttttcccag gaatatgagt caattgaccc tggccagcag ttcacttggg aacattcaaa 3000

cttggaagta aacaaaccaa agaatagata cgcgaatgta atcgcatatg atcattcccg 3060

ggttctccta tcagctatag aagggatccc aggaagtgac tatgtgaatg ccaactacat 3120

agatgggtat aggaagcaaa atgcctatat tgcaacacag ggatctctcc ccgaaacatt 3180

tggggacttt tggagaatga tatgggaaca acggagtgcc acagttgtca tgatgacaaa 3240

actagaagaa agatcaaggg tgaagtgtga ccagtattgg cctagcagag gcacagaaac 3300

ccacggactc gttcaagtaa cgctgcttga tactgtggag ctggccacat attgtgttcg 3360

aacatttgca ctttacaaga atggttcaag tgagaagaga gaagtgagac aattccagtt 3420

caccgcctgg cctgatcatg gtgttccaga acaccctaca ccttttctag ctttcttacg 3480

tagagtcaaa acctgtaacc ctcccgatgc tggtccgatg gttgtgcact gcagtgcggg 3540

agttggccgg actggttgct tcatcgtcat agatgccatg ttagaaagaa taaagcatga 3600

aaaaactgta gatatttatg gccatgtaac tttaatgaga gcccagagga actatatggt 3660

tcaaacagaa gaccaataca tctttatcca tgatgcactg ttagaagcag tgacttgtgg 3720

aaataccgaa gtgccagcta gaaacttgta tgcctacatt cagaagctga cacaaataga 3780

aacgggagag aatgtcacag gaatggagct cgaatttaag cgtctagcca gctcaaaagc 3840

tcacacctca aggtttatca gtgccaatct tccatgtaat aaattcaaaa atcgccttgt 3900

taatattatg ccatatgaat ccacaagggt atgcctgcag cctatccgtg gagtagaagg 3960

atctgattac atcaatgcca gttttattga tggatacaga caacagaaag cctacatcgc 4020

tacccagggg cccttggcag agaccactga agacttctgg cggatgctct gggaacacaa 4080

ttccaccata gttgtgatgc tcaccaagct gcgtgaaatg ggcagagaga aatgtcacca 4140

atactggcca gcagaacggt ctgcaagata ccagtacttt gttgtagatc ccatggctga 4200

gtacaacatg ccacagtata tcctaaggga attcaaggtc acagatgcca gggacggcca 4260

gtcccgaaca gtaaggcagt tccagttcac tgactggcca gagcaaggag tgccaaagtc 4320

cggagaagga tttattgact tcatcggcca agtccataaa acaaaagaac agtttggcca 4380

agatggaccc atttcagtcc attgcagcgc gggcgttgga agaactggag tcttcataac 4440

gctaagcatt gttttggaaa gaatgagata tgaaggagtt gtagatatct tccagactgt 4500

caaaatgtta agaacacaac gaccagctat ggtacagaca gaggatcaat atcagttttc 4560

ctatcgtgcc gcactagagt acctgggcag ctttgaccac tatgcaacgt agaaacccct 4620

gacccattct ggatttttac tacaggccct tcaatatcca tggagtctct tctgagccat 4680

acagggcact tgagaagtcc ttcttaactt ctagctaaca actacttagt gggactatta 4740

cacacaaaac aaattaaaaa caaattattc caggtggacc aagaattcac agtttaataa 4800

tctaacctga aaaataatga agagaatcct gactgatgag gcatctgaag gattttattt 4860

acagatacct caaggattca aaatcaaggg aagaagaaat cacagcaaag aaccaccatc 4920

ttattcagga ttgcttgaaa ggtgcaagga gaaattgcca gaatgctgca gttcagcaca 4980

agatatgtgc tggtgtggct tctcacaatg aaacggactc tgctttgaca tcgccccttc 5040

ccaccatact gctcataata acattttagg gggaaagggg agggaatgtt taaaaagaaa 5100

gtccttgatt tagtttttta gtattgtaaa gatactgctg acctgtgctt catttctaac 5160

tgtgtaaact tttttttaac aaaatgtatc attcgataaa gtgaatttta aaaaagttac 5220

ataactcttc atttttttat ttccaaatcg taggtttttt taagctgtag aactgttatc 5280

tgtaatatct tgctgtagaa atatcaaatc atttgaaaat ttgcacattt ttgtgcaaaa 5340

ctcaatctac agtatcagtc tcgtcagata tattaatttt acacttcagt tttgattggt 5400

gagaaagtac ccattctctt caaataatca aagataatta ttattttgtt ttgtttttgg 5460

aatcaacagg gaggcgcaaa gtataaagtt gctgctaaca tatatacata tatatccata 5520

ttttataagg gtgtctatgt atatatagac agtgtgtcca cacaaaaaat agatacagtt 5580

atcagtcagt cagttcttcc atgatttagt ttttttaaac gtagaaaagc tattgtaaac 5640

atctctttcc atttattctt aattttttga catattggta tttctttaaa gggaaatgag 5700

gaatgcacat cagtgattga ttgtcaaacc tcaccccctg atttcctacc taatctaccc 5760

ccacctaacc aatcaatcac atccacaaat tgttttgttt gtttgttagt caggcttcca 5820

acaaagttca atatttctaa cactctagtg caataaaaat tattattaaa tagctaagag 5880

gtgtgcatgt gggaaaggtc agtgcatatc cctttaggag gggagaatgt tgtaatatat 5940

cagctatcga gttgtttaaa aaaagtgtat tcaatcgtat attgtctata gtatgtgcta 6000

tgaaatttgc atttatgata tgtaacaggg gcaaagccaa attcatgtta ctctgttcag 6060

tcagaaacat tttgtggcat acagcattcc tgggaagtgc tgtactttgt ttcgttttgg 6120

ttttagtttt gcatttagag tgccttataa ttgatgccta ttttaatagc atttcttttt 6180

agcttttggt tcgtatttcc attcactgtt cgtatctgtt actttctatt aaagcattat 6240

ctgtttacca catgtacaaa aactctttga ataatatgca ttcctagttt tcagccaagt 6300

cggggatgtt agtgattgta ccagcccaaa gcacttggat aatcagggcc cttcttcctt 6360

ttataatcaa tcatcaacat cagaaaaagc tacttgtttt atttatattc ccttccaaat 6420

ccgctctgga acatgcagta actgcaccaa acttatttta gtaacaaata tcattggcaa 6480

ctttggaata tatttgatat tccattagga tttttctaaa aggggaaata aactatatat 6540

atatatgtat cttaccccca attcttccaa cagaatttct ataggaagcc atggatgatg 6600

gcataagttt gccacatatt acatgatttt aaataatcct caaaataccc aaggaactct 6660

taaagagttt tggtatgagt atactacttt ggtttaattt tagcttcatg gatgttctgc 6720

atggaaggat ttttgttttc cacattttcc cattgctagc agagtgaaat ccaagagacc 6780

aaacatttgc aagcattgta tttgagcact tttgtaaaaa acaaagaaaa agaaaaaaaa 6840

gaaaatatat ataatactaa aaaaaagtat ctagaaggct acctcagaat gagactctct 6900

aacctacatc agaaccagag aagaatgtgc actatgtggg tctgttatca ttattttctt 6960

ttagtttgta tctttttgga gatttatcca agtgccagat tactcagtgc tataattttc 7020

ttttagttaa acaaaggggg tcagacagac attgcatcat ccagacatgc cttgttggac 7080

atgtagaatc cgatggagca ctgcacacca gaatgattgg ccaatgagca gcttctctcc 7140

ctgaaacaat aactgcccat ttggcaaagg gaaagatgac aataatcaga agaagaaaat 7200

gaatgggatg cataccatag acgaacgagg cggagactat tgcgggaatc ttactgttca 7260

ggagctgttc ctagaactaa ctcccttact gtcattgatg tgcattccac tctgtgcttt 7320

tctgtacaac cattcaagtt ttaatttccc aggtgaacca tctttatctg ccattaccac 7380

aagctttcaa gtttccagtt attttcatca tcataaccag tacggtgcta ttatttacct 7440

atgtacgtgt agttatgtat aattttgtaa ttagttacaa tggtaaaaaa aatcaaaata 7500

tataaaaagt gatttgtaca gaactttatt ttagctcttt tttaaaaatg atttgcatgg 7560

ttagaaaacg gcgaggacag ccaggggagg gaagggcctc tagggaactt tgcactttct 7620

atacctttgt actatgcact gccctattga ttctacaccc aataatgata ttacttgaac 7680

ccatctgtaa gaaactgctt cggaaattca tttgtgtgta tgtaaataac acaacataga 7740

aacaggaagg gaaaaaagtc tgcagtaatg cacgtatttt ttttctttcc tgtttatttt 7800

cggttttgct ttaagtcctt ttatttttaa ttcccttttt gtttttcttt ttgggttttg 7860

gttccttttg ggtttatggg tgccctgata ctccagcaga gatcagaagg ctacagatcc 7920

attctatcca tccgttatgt ggctttgcca tcccagcttg gagtgtcttt acaaagataa 7980

taacagttgt gttctttgct ctcgttttgg atgcatagac tgaaaaatta aaacaaataa 8040

cttgtaaaat ggcttgttaa aaaatacaat tacctctaat tagtagtacg cgtaaatgtt 8100

ttacagaatg aaaggcgtgc tttttatttt cttacttcgt tacattggtg gcgaaagaag 8160

tctgtatgaa aatcagttct ttgctgacac aagttccatt tgttacaaat gaattctaat 8220

aaaaatgtca gtgttatgca aaaaaaaaaa aaaaaaa 8257

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. a PTPRD 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.PTPRD the application of gene in preparation detection diabetes in situ hybridization test kit.

Images