CN101993921A - In-situ hybridization assay kit and detection method for PLAU gene and application of kit - Google Patents

Abstract

The invention relates to an in-situ hybridization assay kit for PLAU gene. The in-situ hybridization assay kit comprises a hybridization probe and a marker, wherein the sequence of the hybridization probe is shown as SEQ ID NO. 1. The invention also provides an in-situ hybridization detection method for the PLAU gene. In addition, the invention also provides application of the kit for preparing medicaments for detecting early transfer of cancers and recurrent diseases. The invention has the advantages that: the provided kit has the characteristics of high sensitivity and high specificity; and the detection method is convenient and easy to operate and can be commonly used and popularized in hospitals of district-level and above.

Description

A kind of hybridization in situ detection kit of PLAU gene and detection method thereof and application

[technical field]

The present invention relates to a kind of test kit, specifically about a kind of hybridization in situ detection kit and detection method and application of PLAU gene.

[background technology]

An annual report has been done by how tame units such as U.S. sanitary research institute in 2005, cancer research institute, Disease Control and Prevention Center, " think that the mankind are failures in anticancer Great War ", that is to say that cancer mortality does not reduce, it lists and causes the Several Factors of anticancer Great War failure to be: 1, tumour cell heterogeneity; 2, tumor cell drug resistance; 3, cancer therapy drug mentality of designing imperfection etc.Simultaneously, also propose to examine closely again the measure of existing diagnosis and treatment cancer in this report.The inventor finds that under study for action two major reasons in addition that cause cancer mortality not fallen are: 1, can not accomplish real early diagnosis; 2, the pathomechanism of Zhuan Yiing is unclear.Come diagnosing cancer according to traditional medical image and with other biochemistry (as protein marker) index, think that occupancy cancer piece is the diagnosis (littler asymptomatic sometimes sign) that belongs to early-stage cancer under 2 centimeters, this clinical concept is worth conscientiously discussing.It is rigorous inadequately that 2 centimeters early stage these of following cancer pieces genus of medical imaging define science, from the cytology angle, 1 centimeter lump has 100,000,000 tumour cells approximately, its three-dimensional cell stack number of 2 centimeters lump is far above 200,000,000 tumour cells, produce and form 2 centimeters cancer piece to the mono-clonal cancer cells early stage from canceration, its pathology evolution process is quite long, may be more than 1 year or 2 years even 3 years, what be difficult to confirm is in this process, and lump is unique spot of cancer and independent focus.Confirm clinically: in case when forming lump, other cancer cells are moved to other position clonal growths by different approaches; In case behind the excision primary tumor, other organ recurrence kitchen ranges or multiple cancer piece kitchen range successively form or shift.Therefore, whether define in early days rigorous inadequately (some case clinically with the lump size below 2 centimeters, when finding primary lesion, find metastatic lesion simultaneously, not in the content of our statement), at this moment be late period, this is the true cause that causes cancer mortality not fallen.

Urokinase type plasminogen activator system, its composition comprises urokinase type plasminogen activator (urokinase type plasminogen activator, uPA or PLAU urokinase gene).The former activation by the tissue protein B in the blood plasma of uPA that studies have shown that tumor cell secretion is double-stranded activated uPA, and latter's plasminogen activation is converted into plasmin.And the plasmin that generates feeds back and activates that uPA is former to be converted into activated double-stranded uPA, produces more activated uPA, and plasminogen activation forms more plasmin more then, to adapt to the needs of tumor cell invasion, transfer.Have and studies show that in a large number the grade malignancy of uPA and acceptor uPAR expression level and kinds of tumors, prognosis and Invasion and Metastasis ability are closely related, can be used as the prediction index that tumor invasion shifts.Discoveries such as Kaneko, the expression of uPA is with tumor invasive depth, tumour cell differentiation, the cancer of the stomach lymph vessels is invaded and microvessel density (MVD) is relevant in cancer of the stomach, the expression of uPAR is invaded relevant with tumour size, invasive depth, the differentiation of nodus lymphoideus transferring rate tumour cell and blood vessel, show that through multiplicity the expression of uPA can be used as the prognosis of gastric cancer index in the cancer of the stomach.PLAU (urokinase) gene, NM_002658,2395bp, mRNA, 10q24 ", cds:147 ... 1442bp.

Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as cancer genomics, so far, we might do more accurate early diagnosis on gene level, form (during mono-clonal) at canceration precancer cell and just can accomplish the early prediction diagnosis.It was that hybridization technique detection UPA expression of gene amount is diagnosed clinical all kinds of cancer originally that the present invention adopts nucleic acid, and extremely important clinical value is arranged.

Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, 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.

[summary of the invention]

The objective of the invention is provides a kind of purposes of hybridization in situ detection kit of PLAU gene at deficiency of the prior art.

One purpose more of the present invention is that a kind of hybridization in situ detection kit of PLAU gene is provided.

Another purpose of the present invention is that a kind of in situ hybridization detection method of PLAU gene is provided.

For achieving the above object, the technical scheme taked of the present invention is:

A kind of hybridization in situ detection kit of PLAU gene detects cancer in preparation and shifts, recurs application in the disease medicament in early days, and described test kit comprises hybridization probe, marker, and described hybridization probe sequence is shown in SEQ ID NO.1.

Described cancer is a cancer of the stomach.

The RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

Described marker is selected from radionuclide or non-radioactive marker.

Described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

Described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

Described non-radioactive marker is preferably from digoxin.

Described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

A kind of hybridization in situ detection kit of PLAU gene comprises hybridization probe, marker, and wherein said hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is:

A kind of in situ hybridization detection method of PLAU gene, this method may further comprise the steps:

A, the hybridization probe in the test kit is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein Za Jiao concrete steps comprise:

Instrumentation:

1). sample to be measured is put into reactive tank;

2). instrument discards liquid automatically, adds Digestive system automatically;

3). instrument discards liquid automatically, and the back is fixing automatically;

4). instrument discards liquid automatically, automatically prehybridization (42 ℃);

5). instrument discards liquid automatically, cleans automatically;

6). instrument discards liquid automatically, automatically hybridization (42 ℃);

7). instrument discards liquid automatically, cleans automatically;

8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);

9). instrument discards liquid automatically, cleans colour developing automatically;

10). take out the mounting microscopy.

The invention has the advantages that:

1, test kit provided by the invention has characteristics highly sensitive, high specificity.

2, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.

3, diagnostic kit of the present invention and other detection oncofetal protein mark clinically, and the medical imaging inspection has remarkable difference.The present invention can detect the PLAU gene unconventionality expression on gene level, before the recurrence of occupancy carninomatosis kitchen range is not found in the medical imaging inspection, before the cancer biochemical indicator does not produce unusually, also do not form before the tumour, can accomplish the information acquisition of above abnormal gene expression early, predict for real early diagnosis of clinical cancer sufferer and treatment back transfer and relapse early.So just might implement early diagnosis, early prevention, the early treatment of cancer, might from the source, thoroughly effect a radical cure the cancer foul disease.

[description of drawings]

Fig. 1 is a Metastasis of Gastric Cancer patient P LAU genetic expression picture in the embodiment of the invention.

Fig. 2 is a normal people PLAU genetic expression picture in the embodiment of the invention.

[embodiment]

Below in conjunction with accompanying drawing the specific embodiment of the invention is elaborated.

Embodiment 1

A kind of hybridization in situ detection kit of PLAU gene comprises hybridization probe, marker, synergistic agent, and wherein, described hybridization probe sequence is shown in SEQ ID NO.1.The hybridization probe digoxigenin labeled.Other liquid and sample in the test kit are composed as follows:

Digestive system 100 μ l/ manage 1 pipe/box colourless transparent liquid

Protection liquid 100 μ l/ pipe 1 pipe/box colourless transparent liquid

Prehybridization solution 1300 μ l/ manage 2 pipe/box colourless transparent liquids

Justice hybridization solution 10 μ l/ pipe 1 pipe/box colourless transparent liquid

Antisense hybridization solution 10 μ l/ manage 1 pipe/box colourless transparent liquid

Confining liquid 1000 μ l/ manage 1 pipe/box colourless transparent liquid

Alkaline phosphatase enzyme antibody 1 μ l/ manages 1 pipe/box colourless transparent liquid

Developer A 175 μ l/ manage 1 pipe/box yellow liquid

Developer B 320 μ l/ manage 1 pipe/box colourless transparent liquid

Light yellow or the colourless transparent liquid of damping fluid I 10x 90ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid II 10x 80ml/ bottle 1 bottle/box

Light yellow or the colourless transparent liquid of damping fluid III 10x 20m/ bottle 3 bottle/boxes

Light yellow or the colourless transparent liquid of damping fluid IV 10x 90ml/ bottle 1 bottle/box

Stationary liquid 90ml/ bottle 1 bottle/box colourless transparent liquid

6/box of positive control sample

Mentioned reagent composition explanation: (all reagent are available from SIGMA)

1, Digestive system: the 20mg/ml Proteinase K, the 100mg Proteinase K adds DEPC-H ₂O 5ml;

2, the glycine of protection liquid: 0.2g adds 1 * damping fluid I of 1ml;

3, prehybridization solution: 1 * damping fluid II7.5ml

50×D?3ml

10mg/ml?yest?t-RNA?750ul

11mg/ml?SALMON?TESTES?DNA?682ul

0.04M?EDTA?3ml

50％formamide?15ml

4, the bloking of confining liquid: 0.03g (buying from Roche Holding Ag) adds 1ml 1 * damping fluid III;

5,10x damping fluid I:(PH7.1-7.4)

NaCl?80g

Na ₂HPO ₄.12H ₂O?360g

KCl 2g

KH ₂PO ₄2g

Add tri-distilled water to 1l, and autoclaving;

6,10x damping fluid II:(PH7.0)

NaCl 175.3g

Trisodium Citrate 88.2g

Several of HCl

Add tri-distilled water to 1l, and autoclaving;

7, damping fluid III:(PH7.9)

Tris 121.1g

NaCl 87.66g

About HCl 60ml

Add tri-distilled water to 1l, and autoclaving;

8, damping fluid IV:

1M Tris-HCl (PH9.5): Tirs 121.1g adds about HCl 3ml, adds water 900ml, transfers PH to 9.5, adds water to 1l, and autoclaving;

1M NaCl:NaCl 58.44 adds water to 1l, and autoclaving;

0.5M MgCl ₂: 101.65g MgCl ₂.6H ₂O adds water to 1l, and autoclaving;

9, stationary liquid: Paraformaldehyde 96 40g adds 1 * damping fluid I to 1l, and heat (about 50-60 degree) is stirred to dissolving a little;

10, developer A:NBT 1g adds 70%DMF11.44ml;

11, developer B:BCIP 1g adds 100%DMF30ml.

Test kit of the present invention can many person-portions use or person-portion use.

Embodiment 2

A kind of PLAU gene hybridization in situ detection method and test kit thereof are used

One, sample disposal

1, with the centrifuge tube of 10ml, dress 4.5ml lymphocyte separation medium, again the 3ml anticoagulation is slowly added contain lymphocyte separation medium (blood: in centrifuge tube lymphocyte separation medium=1: 1.5), the centrifugal 10min of 2000r/min;

2, draw the middle layer white corpuscle to another centrifuge tube, in this pipe, add 1 * damping fluid I of about twice again, mixing, the centrifugal 10min of 1500g/min;

3, abandon supernatant. precipitation adds 1 * damping fluid I of about twice, mixing, the centrifugal 10min of 1500g/min;

4, abandon supernatant, and test tube mouth excess liquid is gone with the tissue suction.Again precipitation is made suspension, drop in push jack on the slide, seasoning.(hospital with good conditionsi can use the pelleter film-making.) 3ml blood, can do 4 slice, thin pieces;

5, with 40ml 4% stationary liquid, in glass jar, fixedly 30min uses 1 * damping fluid I to wash 5min again.Every cylinder can be put 16;

6, sample can be kept at-20 ℃, or continues to do experiment.

Two, reagent in the test kit is mixed with working concentration

1, with 10 * damping fluid I with tri-distilled water by being diluted to 1 * damping fluid I at 1: 10;

2, with 20 * damping fluid II with tri-distilled water by being diluted to 2 * damping fluid II at 1: 10;

By being diluted to 0.2 * damping fluid II at 1: 100; By being diluted to 0.1 * damping fluid II at 1: 200;

3, with 10 * damping fluid III with tri-distilled water by being diluted to 1 * damping fluid III at 1: 10;

4,10 * damping fluid IV with tri-distilled water by be diluted at 1: 10 * damping fluid IV (get 1#, 2#, each 10ml of 3#, add water to 100ml both can).

Three, experimental procedure:

1, gets two of every person's samples to be checked, two of (other two give over to check with) and positive control samples (test at every turn and do a pair of positive control);

2, in glass jar, add Digestive system (Digestive system 100ul adds 1 * damping fluid 199.9ml, is working concentration) 20ml.37 ℃ of water-bath preheatings 10 minutes.Put 16 slides into, handle 12min, use 1 * damping fluid I to wash 5min again for 37 ℃;

3, wash 10min with 0.2% protection liquid (protection liquid 1ml adds 1 * damping fluid I99ml and is working concentration), tri-distilled water is washed 5min, and above process is all carried out at glass jar.The slide seasoning;

4, slide is put into the box of preserving moisture, add prehybridization solution 20ul/ sheet. covered, the lid box of tightly preserving moisture 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, with 70%, 90%, 95% ethanol is respectively washed 2min, seasoning;

6, slide is put into the box of preserving moisture, two of every patient specimens, one adds just hybridization solution 20ul/ sheet, and another adds antisense hybridization solution 20ul/ sheet, covered, the lid box of tightly preserving moisture 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, each 15min with 2 * damping fluid II

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, each 15min with 0.1 * damping fluid II;

8, wash 30s with 1 * damping fluid III, take out slide, seasoning;

9, slide is put into the box of preserving moisture, add 0.5%l confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture, at room temperature act on 30min;

10, take out slide, III washes 30s with 1 * damping fluid, seasoning;

11, slide is put into the box of preserving moisture, add alkaline phosphatase enzyme antibody (adding 1.8ml 1 * damping fluid III) 100ul/ sheet, cover the box of tightly preserving moisture and at room temperature act on 30min;

12, take out slide, wash 3 times, each 15min with 1 * damping fluid III;

13,1 * damping fluid IV washes 2min, adds developer (developer A73.3ul, developer B157.5ul are added among 30ml 1 * damping fluid IV, mixing), more than the room temperature lucifuge 12h;

14, wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.

Four, the result judges

100-300 cell of counting calculates the per-cent of catching the purple cell under light microscopic.

What the positive control sample added the antisense hybridization solution should catch purple more than 80%.

All add the negative internal reference of just hybridization solution should be colourless.

The cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also having overcome biotin labeled probe is organized the endogenous vitamin H to do 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 mRNA,, judge the expression amount of goal gene according to painted cell count.

The inventive method is a nucleic acid hybridization in situ technology at present commonly used, and this method is by detecting the PLAU gene expression amount in the substrate cell, is used for determining whether cancer takes place and/or shift.

Clinical study shows that the PLAU gene has broad spectrum, because the PLAU gene is expressed low in the normal people or zero expression.If the PLAU gene high expression illustrates that cancer recurs, shifts, spreads, thus the diagnostic message of acquisition cancer.When detecting the PLAU expression of gene and be higher than normal control, to be that cancer is early stage shift or the cancer metastasis susceptible person then measurable experimenter, and these cancers comprise cancer of the stomach.

The embodiment of the invention is sampled as: 5 of Metastasis of Gastric Cancer patients, 5 of normal control 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 cancer metastasis patient P LAU genes have overexpression, cell dyeing; Normal control group PLAU gene is not expressed the cell dye-free.Concrete outcome is asked for an interview Fig. 1, Fig. 2.

Embodiment 3

Detect the Metastasis of Gastric Cancer disease and detect parallel laboratory test between the Metastasis of Gastric Cancer disease with the PLAU kit gene with the CA125 kit gene.

Specificity, susceptibility, accuracy for each comfortable Metastasis of Gastric Cancer disease of scientific evaluation said gene.We use the method for parallel test, detect the mRNA of said gene simultaneously, detection technique adopts the nucleic acid hybridization in situ technology, with same routine Metastasis of Gastric Cancer disease peripheral blood of patients, detect PLAU gene and CA125 (CA125 (tumor marker), NM-024690) mRNA of gene (carrying out same procedure and step and reagent that the hybridization in situ technique of embodiment 1 and embodiment 2 is all adopted in nucleic acid hybridization in situ, immunohistochemical staining, mirror numeration down, report as a result etc.) simultaneously.Find the PLAU gene in Metastasis of Gastric Cancer disease patient expression amount than the expression amount height of CA125 gene same disease patient.The result shows that specificity, susceptibility, the accuracy of the medical diagnosis on disease of PLAU gene pairs Metastasis of Gastric Cancer are better than CA125 gene, and in situ hybridization genetic expression figure shows that PLAU expression of gene amount is 70%, and CA125 expression of gene amount is 45%.The index that test kit of the present invention is done in the Metastasis of Gastric Cancer medical diagnosis on disease has very important clinical meaning.

SEQUENCE?LISTING

＜110〉Rui bends biotechnology (Shanghai) Co., Ltd.

＜120〉a kind of hybridization in situ detection kit of PLAU gene and detection method thereof and application

<130>/

<160>1

<170>PatentIn?version?3.3

<210>1

<211>2395

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

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tcctggtcgt?gagcgactcc?aaaggcagca?atgaacttca?tcaagttcca?tcgaactgtg 240

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Claims (11)

1. the hybridization in situ detection kit of a PLAU gene detects cancer in preparation and shifts, recurs application in the disease medicament in early days, and described test kit comprises hybridization probe, marker, and described hybridization probe sequence is shown in SEQ ID NO.1.

2. application according to claim 1 is characterized in that: described cancer is a cancer of the stomach.

3. application according to claim 1 is characterized in that: the RNA sequence of described hybridization probe sequence shown in SEQ ID NO.1.

4. according to the arbitrary described application of claim 1-3, it is characterized in that: described marker is selected from radionuclide or non-radioactive marker.

5. application according to claim 4 is characterized in that: described radionuclide is selected from ³H, ³⁵S, ¹²⁵I or ³²A kind of among the P.

6. application according to claim 4 is characterized in that: described non-radioactive marker is selected from a kind of in vitamin H, digoxin, alkaline phosphatase, horseradish peroxidase or the fluorescein.

7. application according to claim 6 is characterized in that: described non-radioactive marker is preferably from digoxin.

8. according to the arbitrary described application of claim 1-3, it is characterized in that: described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

9. the hybridization in situ detection kit of a PLAU gene comprises hybridization probe, marker, it is characterized in that, described hybridization probe sequence is shown in SEQ ID NO.1, and described marker is selected from radionuclide or non-radioactive marker.

10. the in situ hybridization detection method of a PLAU gene is characterized in that, this method may further comprise the steps:

A, the hybridization probe in the described test kit of claim 9 is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

11. detection method according to claim 10 is characterized in that: the condition that forms hybridization complex in a step is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour, and described substrate is selected blood cell sample or histocyte sample for use.

Images