CN101993935A - In-situ hybridization assay kit and detection method for MTDH gene and application of kit - Google Patents

Abstract

The invention relates to an in-situ hybridization assay kit for MTDH 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 MTDH gene. In addition, the invention also provides application of the kit for preparing medicaments for detecting breast cancer. 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 MTDH 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 MTDH gene

[background technology]

Mammary cancer is one of modal malignant tumour of women, and according to statistics, sickness rate accounts for the 7-10% of the various malignant tumours of whole body, is only second to uterus carcinoma the women.Its morbidity is normal relevant with heredity, and between 40-60 year, climacteric, women's sickness rate of front and back was higher.Only the mammary gland patient of about 1-2% is the male sex.Usually occur in the malignant tumour of breast glandular epithelium tissue.Be one of a kind of women's of having a strong impact on physical and mental health even life-threatening modal malignant tumour,

Mammary cancer changes to the formation cancer before cancer, need several years, how to accomplish early detection, diagnosis, prognosis detection and the recurrence after the diagnosis and treatment, shift that to detect in early days be the key that reduces M ﹠ M.

MTDH (METADHERIN) gene works in tumour takes place and shifts, and in clinical all kinds of tumours high expression level is arranged all.American Studies personnel claim, they find that the MTDH gene plays a crucial role in patient with breast cancer's body, and it has increased the probability of cancer cells diffusion and can resist chemotherapy, and the correlative study achievement is published on " cancer cells " magazine of publishing January 5.Stop the cancer cells diffusion quite important, studies show that, do not have to survive more than 5 years above 98% patient, and in the patient that cancer cells has spread, have only 27% people can survive several years among the patient of diffusion at cancer cells.The Mi Xieer of New Jersey cancer research institute. the gene of tumour diffusion has been adopted several method to seek to make in Leixs etc., they have called huge tumour cell database, discovery is suffered from the patient body of serious mammary cancer, and the sub-fraction of its No. 8 human chromosomals has been repeated repeatedly.In general, a gene of most of normal DNA sequences only has 2 copies, but the copy of this part patient's gene reaches 8.Then, the researchist has analyzed the mastocarcinoma gene sample that picks up from 250 patients, to seek heritable variation wherein.Discover, in being rich in the tumour of aggressivity, its MTDH gene overacfivity or by overexpression, this gene can not only make tumour cell tightly on the blood vessel attached near the organ diseased region, and the effect that can also weaken cancer therapy drug greatly makes tumour cell produce resistance.The researchist then injected on one's body in experimental mouse and picked up from having this genovariation patient's tumor cell, found to have occurred in the mouse body can diffusion tumour, these tumours have also suppressed the result of treatment of traditional chemotherapeutics such as taxol.But after the researchist carried out genetic engineering to these tumours, has suppressed the MTDH gene, tumour cell no longer spread and chemotherapy is no longer included resistibility.The researchist says that MTDH may also play a major role in comprising many other class cancers of prostate cancer.The researchist wishes, their discovery will help to develop and can not only stop the tumour cell diffusion and to the therapeutic response better medicament.In similar research, also find MTDH protein gene and 8q22 genomic locus be two with the existing clinical marker of mammary cancer (as ER, HER2 etc.) weak prognostic markers fully independently, can consider to develop into the prognosis test kit, be used for finding as early as possible the excessive risk patient of needs diffusion treatment.

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, just can accomplish the early prediction diagnosis in canceration early stage or cancer cells formation (during mono-clonal).The present invention adopts nucleic acid hybridization in situ technology for detection MTDH expression of gene amount, and the diagnosis of the early gene level of breast cancer is had very important clinical meaning.

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 MTDH 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 MTDH gene is provided.

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

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

The application of a kind of hybridization in situ detection kit of MTDH gene in preparation detection breast cancer disease medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence such as SEQ ID:MTDH gene, MTDH sequence number: NM-1788127667bp CDS:329 ... 2077bp is at karyomit(e) 8q22.1 ".

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 MTDH 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 MTDH 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, clinical meaning of the present invention is that more early stage tracking detects mammary cancer generation dynamic process, and the detection and the screening implement that are used for the mammary cancer preventive medicine.Diagnostic kit of the present invention is with other detects the oncofetal protein mark clinically, and the medical imaging inspection has remarkable difference.The present invention can detect the MTDH unconventionality expression on gene level, before medical imaging inspection and other inspection is not found occupancy mammary cancer focus, before the cancer biochemical indicator does not produce unusually, also do not form before the lump, can accomplish the information acquisition of above abnormal gene expression early, give real prognosis early diagnosis of clinical mammary cancer sufferer.So just might implement early diagnosis, early prevention, the early treatment of mammary cancer, might from the source, thoroughly effect a radical cure the mammary cancer foul disease.

[description of drawings]

Fig. 1 is a mammary gland cancer patient MTDH genetic expression picture in the embodiment of the invention.

Fig. 2 is a normal breast MTDH genetic expression picture.

[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 MTDH 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 II 7.5ml

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 MTDH 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 5ml1 * 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 commonly used at present, this method is by detecting the MTDH gene expression amount in the substrate cell, be used for determining whether mammary cancer takes place, because the MTDH gene is low the expression in the normal people, if MTDH genetic expression height, illustrate that cancer takes place, thereby obtain the diagnostic message of cancer.

The embodiment of the invention is sampled as: 5 of mammary 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 patient with breast cancer MTDH genes have overexpression, cell dyeing; Normal control group MTDH gene is not expressed the cell dye-free.Concrete outcome is asked for an interview Fig. 1 and Fig. 2.

Embodiment 3

Detect breast cancer disease and detect parallel laboratory test between the breast cancer disease with the MTDH kit gene with the ERK kit gene.

Specificity, susceptibility, accuracy for each comfortable breast 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 breast cancer disease peripheral blood of patients, detect MTDH gene and ERK (ERK oncogene simultaneously, sequence number NM-002745 mRNA 5916bp CDS:241 ... 1323bp is at chromosome 22 q11.21 ".) mRNA (carrying out same procedure and step and reagent that the hybridization in situ technique of embodiment 1 and embodiment 2 is all adopted in the numeration down of nucleic acid hybridization in situ, immunohistochemical staining, mirror, report as a result etc.) of gene.Find the MTDH gene in the breast cancer disease patient expression amount than the expression amount height of ERK gene same disease patient.The result shows that specificity, susceptibility, the accuracy of the diagnosis of MTDH gene pairs breast cancer disease are better than ERK gene, and in situ hybridization genetic expression figure shows that MTDH expression of gene amount is 70%, and ERK expression of gene amount is 50%.The index that test kit of the present invention is done in the breast cancer disease diagnosis has very important clinical meaning.

The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.

SEQUENCE?LISTING

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

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

<130>/

<160>1

<170>PatentIn?version?3.3

<210>1

<211>7667

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

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gaggctaagg?cagagaatca?tttgaaccca?ggagacggag?tttgcaaagc?caagatcgtg 4440

ccactgcact?ccagcctggg?caacagaagg?agactccgtc?tcaaaaaaaa?aaaaaagatg 4500

gcagctatat?aaatgataaa?attaattaca?ttctctttca?catgcatgag?gtgcaaactc 4560

tgtcacaaag?tattttaatt?accttttacc?ttgtttcata?gatctttatg?tgacataaaa 4620

acagtttctg?gcacggtggc?tcacgcctgt?aatcctagca?ctttgggagg?ctgaggcagg 4680

tggatcacct?gaggtcagga?gtttgagacc?agcctggcca?atatggtgaa?accccatctc 4740

tacaaaattt?gcaaaaagta?gatgggtgtg?gtagtgggcg?cctgtaatcc?cagctactca 4800

ggaggttgag?gcagagaatc?gcttgaaccc?ggggggtgga?ggttgcagtg?agctgagatc 4860

gcaccactgc?actccagcat?gaaagagcga?gactcaatct?caaaaaaaaa?aaagtttctg 4920

gcacctgaac?aggaactggt?ttccatcatc?aactcagaaa?gcactaaaat?ctaggtggtg 4980

attcagggag?gagcagggga?agacagcctc?ctatggtggc?atgaataaga?tgcttccaga 5040

actagtaggg?aaataactaa?cctcttcagg?ctttatcagg?cctggagggg?aaccttgctc 5100

atgttagcaa?gaaaggtatc?ctagagaagc?cactcaaaag?gctccctaat?ccagcctgtc 5160

tccacataca?tactgaaaat?tcttccctac?tctgaggcag?ggtgtagtgg?tttaggggtt 5220

tctccagact?ggaatcctac?ctatctgtac?cgacaattga?gcaaacaaca?gttgagagag 5280

tccaaaaaaa?aaagtattaa?aatgtgattg?atgtaattta?ccatgtttac?tttatgcatg 5340

cattttattg?gggaggggag?gtcagaataa?ttcacccaaa?tctagtggtc?ttatttcata 5400

ggctaatctg?gtttatattt?gcattaaaga?tactggaggg?caatatttac?agagtttagt 5460

ttttcttaat?taaaaacagt?cctctattaa?tatagtgtga?aatatctttc?aaaatttaga 5520

gtttaggttt?aagatgtcta?ctagatatct?ttaagatttt?cctgtaaact?cactgcacaa 5580

actggaatta?ctttccaaaa?gacttaggga?atgcaaatat?gttactcata?agatgcattg 5640

agtattgtaa?ataaaacaaa?ccatttttga?tttgtttaaa?ttgctcgtta?cagttctctt 5700

gtggggaggg?actttgtcag?tcattttgca?tcttaagcta?gactaaactt?tttgttgttg 5760

ttttcctaaa?accataggtg?caagctttgc?cgctgggaag?tcatattgaa?ttaagcactt 5820

ttgaaaatgt?cactgtttgt?gacacacaat?gttctctaca?gaaaacttct?tctatcttat 5880

gtcattttag?cagtagatgt?agctgcctgc?caccaggtgc?aatatgtcat?ttgcatcagc 5940

cttttcagtt?aaggaaatta?aaacttggca?tgcattagtt?catatgtatg?aggttggctg 6000

tgtccccaaa?cagcaattgc?ttgtacaaga?tagaagtttg?cttctcagct?gggcatggtg 6060

gctcatgcct?gtaatccaag?ctctttggga?ggccaacgcg?ggaggattgt?ttgagcccag 6120

gagtttgaaa?ccatcctgag?caatagagag?acccccatct?cgacaaaaaa?aaaaaaaaaa 6180

aattagaaac?aaaaaaaaga?ttgtttctct?ttcatattaa?agtttgggaa?ggcaatccag 6240

agcccatatg?acattatgca?gggtcaggga?tccgggttct?ttctatattg?ttcctcccca 6300

ttctcaacct?gtagcttttt?ttttttttct?tttaatgaga?tagggtctca?ctctgttgcc 6360

caggctggag?tgcagtggca?cacttctggc?tcacttcagc?ctggaactcc?cgggctcgag 6420

ctatattctc?ccactttagc?ctcagcctcc?agagtacctg?ggactacagg?tacacaccac 6480

catgcctggc?taaatttttt?tttttttttt?ttggtagaga?tgaggtcatt?gctatgttgc 6540

ccaggctggc?cttgaagtcc?agggcttaag?tgatcctcct?gccttggcct?cccaaagtgc 6600

tggtgttaca?ggtgtgagcc?accacacctg?gccaacatgt?ggctttcagt?tcttacttga 6660

agacagctgc?tctagctaga?aacttcctat?cggcatctga?gccagctggt?agagggcatt 6720

acctccccca?cccccaagaa?cacttcttgg?aagttacaca?caatacataa?acttcaattt 6780

caaacagttg?cttggtccca?ctagccatga?agctggatgg?gacgtaatat?tctatctggg 6840

ttgtcatggg?ccaaattaac?attttgagat?tctgttagga?agatgagaat?tggataatgg 6900

gcacctctaa?cagtgtctgt?cagagttgac?atacattgtg?tatctcctgc?accaaagcat 6960

ttttgagatc?aagtgttctt?ccagctgagc?agaaatttgg?aaggctattc?agtgctgctt 7020

agtgtagcag?ctaataatgt?tccaacttct?atatgatagt?ttgctttttg?ttttttgttt 7080

ttttctagag?acagagtctc?gttgcccagg?ctggtctcca?attcctgggt?tcaagcaatc 7140

ctcctgactc?agcctcctaa?atgctgggat?tacaggcatg?aaccactgtg?cccagtctat 7200

atataataga?ttttagaaga?aatttctgcc?actcagtgac?tgctttaaat?tctagagaca 7260

gaggctgaga?gaaacttagt?agcctgcctg?cgcatactgc?aagtacagac?tatataacaa 7320

gttgaaagag?aatcaccctg?gtatataata?ttttaaaaca?tgaaaaaggc?attactattg 7380

ttctggtcta?agtactctaa?caggacgatg?atttctaacc?ctagcatcat?gacatgtata 7440

tagtgtgttg?taagacattt?gccagctaat?agttacagta?ctgaagcttg?tgaatacgtt 7500

aattgagaaa?actttaagca?ggttcaaagt?tatcccatga?tagtggcatt?cttatttgta 7560

tatatgtgct?ttcctttttt?atttgtatca?aggcatttaa?aatgaatata?tgtaacatgt 7620

acctaagttt?taaaagcata?ataaaatgaa?cacatctgaa?accacca 7667

Claims (10)

1. the application of the hybridization in situ detection kit of a MTDH gene in preparation detection breast cancer disease medicine, described test kit comprises hybridization probe, marker, described hybridization probe sequence is shown in SEQ ID NO.1.

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

3. application according to claim 1 and 2 is characterized in that: described marker is selected from radionuclide or non-radioactive marker.

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

5. application according to claim 3 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.

6. application according to claim 5 is characterized in that: described non-radioactive marker is preferably from digoxin.

7. application according to claim 1 and 2 is characterized in that: described test kit also comprises synergistic agent, and described synergistic agent is the alkaline phosphatase enzyme antibody.

8. the hybridization in situ detection kit of a MTDH 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.

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

A, the hybridization probe in the described test kit of claim 8 is contacted with RNA to be measured in the substrate, form hybridization complex;

The hybridization complex that b, detection a step obtain.

10. detection method according to claim 9 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.

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