CN101993941A - In situ hybridization detection kit of CD44 gene and detection method and application thereof - Google Patents
In situ hybridization detection kit of CD44 gene and detection method and application thereof Download PDFInfo
- Publication number
- CN101993941A CN101993941A CN 200910056178 CN200910056178A CN101993941A CN 101993941 A CN101993941 A CN 101993941A CN 200910056178 CN200910056178 CN 200910056178 CN 200910056178 A CN200910056178 A CN 200910056178A CN 101993941 A CN101993941 A CN 101993941A
- Authority
- CN
- China
- Prior art keywords
- hybridization
- gene
- marker
- kit
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an in situ hybridization detection kit of a CD44 (cancer antigen 44) gene, which comprises a hybridization probe and a marker, wherein the sequence of the hybridization probe is as shown by SEQ ID NO.1. The invention also provides an in situ hybridization detection method of the CD44 gene. Besides, the invention also provides application of the kit in preparing a medicament for detecting mammary cancer diseases. The invention has the following advantages that: the kit has the characteristics of high flexibility and strong specificity. The detection method of the invention is convenient and simple to operate, and can be commonly used and popularized in hospitals above district level.
Description
[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 CD44 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.
CD44 is a cytokine, expresses in human primary breast cancer and reduces, and also expresses in other cancer and reduces.Search time to CD44 is long, more deep.Known its is distribution cell surface transmembrane glycoprotein very widely, mainly participates in heterotypic adhesion, i.e. the adhesion of tumour cell and host matrix, and heterotypic adhesion plays a driving role in tumor cell invasion shifts.The target protein major function of CD44 is: (1) mediation lymphocyte combines with high column endotheliocyte in (microcirculation) PCV, makes lymphocyte pass vessel wall or arrives Lymphoid tissue; (2) participate in lymphocytic activation process; (3) combine with substrate molecule such as hyaluronic acid, ln in the extracellular matrix; (4) can combine with cytoskeletal protein, participate in the cell pseudopodium and form, and relevant with the locomotory movement of cell.
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 CD44 gene, and the early diagnosis of mastocarcinoma gene level is had very important clinical meaning.CD44 gene order number: NM-000610,5748bp, CDS:435 ... 2663.
Hybridization in situ technique of the present invention (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 CD44 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 CD44 gene is provided.
Another purpose of the present invention is that a kind of in situ hybridization detection method of CD44 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 CD44 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.
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 CD44 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 CD44 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 CD44 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 CD44 genetic expression picture in the embodiment of the invention.
Fig. 2 is a normal breast CD44 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 CD44 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 CD44 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, and this method is used for determining the prognosis of mammary cancer by detecting the CD44 gene expression amount in the substrate cell.Clinical study shows, the CD44 gene is low the expression in mammary cancer, because the CD44 gene is at normal people's high expression level, the low expression explanation prognosis of breast cancer of CD44 gene is poor, and CD44 expression of gene degree is relevant with the prognosis of breast cancer, belongs to negative correlation, expresses that more to hang down prognosis poorer.Thereby obtain the diagnostic message of mammary cancer.As mentioned above, when detecting CD44 genetic expression, measurable experimenter is the Prognosis in Breast Cancer situation.Concrete outcome is asked for an interview Fig. 1 and Fig. 2.
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 CD44 gene and detection method thereof and application
<130>/
<160>1
<170>PatentIn?version?3.3
<210>1
<211>5748
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>1
gagaagaaag?ccagtgcgtc?tctgggcgca?ggggccagtg?gggctcggag?gcacaggcac 60
cccgcgacac?tccaggttcc?ccgacccacg?tccctggcag?ccccgattat?ttacagcctc 120
agcagagcac?ggggcggggg?cagaggggcc?cgcccgggag?ggctgctact?tcttaaaacc 180
tctgcgggct?gcttagtcac?agcccccctt?gcttgggtgt?gtccttcgct?cgctccctcc 240
ctccgtctta?ggtcactgtt?ttcaacctcg?aataaaaact?gcagccaact?tccgaggcag 300
cctcattgcc?cagcggaccc?cagcctctgc?caggttcggt?ccgccatcct?cgtcccgtcc 360
tccgccggcc?cctgccccgc?gcccagggat?cctccagctc?ctttcgcccg?cgccctccgt 420
tcgctccgga?caccatggac?aagttttggt?ggcacgcagc?ctggggactc?tgcctcgtgc 480
cgctgagcct?ggcgcagatc?gatttgaata?taacctgccg?ctttgcaggt?gtattccacg 540
tggagaaaaa?tggtcgctac?agcatctctc?ggacggaggc?cgctgacctc?tgcaaggctt 600
tcaatagcac?cttgcccaca?atggcccaga?tggagaaagc?tctgagcatc?ggatttgaga 660
cctgcaggta?tgggttcata?gaagggcacg?tggtgattcc?ccggatccac?cccaactcca 720
tctgtgcagc?aaacaacaca?ggggtgtaca?tcctcacatc?caacacctcc?cagtatgaca 780
catattgctt?caatgcttca?gctccacctg?aagaagattg?tacatcagtc?acagacctgc 840
ccaatgcctt?tgatggacca?attaccataa?ctattgttaa?ccgtgatggc?acccgctatg 900
tccagaaagg?agaatacaga?acgaatcctg?aagacatcta?ccccagcaac?cctactgatg 960
atgacgtgag?cagcggctcc?tccagtgaaa?ggagcagcac?ttcaggaggt?tacatctttt 1020
acaccttttc?tactgtacac?cccatcccag?acgaagacag?tccctggatc?accgacagca 1080
cagacagaat?ccctgctacc?actttgatga?gcactagtgc?tacagcaact?gagacagcaa 1140
ccaagaggca?agaaacctgg?gattggtttt?catggttgtt?tctaccatca?gagtcaaaga 1200
atcatcttca?cacaacaaca?caaatggctg?gtacgtcttc?aaataccatc?tcagcaggct 1260
gggagccaaa?tgaagaaaat?gaagatgaaa?gagacagaca?cctcagtttt?tctggatcag 1320
gcattgatga?tgatgaagat?tttatctcca?gcaccatttc?aaccacacca?cgggcttttg 1380
accacacaaa?acagaaccag?gactggaccc?agtggaaccc?aagccattca?aatccggaag 1440
tgctacttca?gacaaccaca?aggatgactg?atgtagacag?aaatggcacc?actgcttatg 1500
aaggaaactg?gaacccagaa?gcacaccctc?ccctcattca?ccatgagcat?catgaggaag 1560
aagagacccc?acattctaca?agcacaatcc?aggcaactcc?tagtagtaca?acggaagaaa 1620
cagctaccca?gaaggaacag?tggtttggca?acagatggca?tgagggatat?cgccaaacac 1680
ccaaagaaga?ctcccattcg?acaacaggga?cagctgcagc?ctcagctcat?accagccatc 1740
caatgcaagg?aaggacaaca?ccaagcccag?aggacagttc?ctggactgat?ttcttcaacc 1800
caatctcaca?ccccatggga?cgaggtcatc?aagcaggaag?aaggatggat?atggactcca 1860
gtcatagtat?aacgcttcag?cctactgcaa?atccaaacac?aggtttggtg?gaagatttgg 1920
acaggacagg?acctctttca?atgacaacgc?agcagagtaa?ttctcagagc?ttctctacat 1980
cacatgaagg?cttggaagaa?gataaagacc?atccaacaac?ttctactctg?acatcaagca 2040
ataggaatga?tgtcacaggt?ggaagaagag?acccaaatca?ttctgaaggc?tcaactactt 2100
tactggaagg?ttatacctct?cattacccac?acacgaagga?aagcaggacc?ttcatcccag 2160
tgacctcagc?taagactggg?tcctttggag?ttactgcagt?tactgttgga?gattccaact 2220
ctaatgtcaa?tcgttcctta?tcaggagacc?aagacacatt?ccaccccagt?ggggggtccc 2280
ataccactca?tggatctgaa?tcagatggac?actcacatgg?gagtcaagaa?ggtggagcaa 2340
acacaacctc?tggtcctata?aggacacccc?aaattccaga?atggctgatc?atcttggcat 2400
ccctcttggc?cttggctttg?attcttgcag?tttgcattgc?agtcaacagt?cgaagaaggt 2460
gtgggcagaa?gaaaaagcta?gtgatcaaca?gtggcaatgg?agctgtggag?gacagaaagc 2520
caagtggact?caacggagag?gccagcaagt?ctcaggaaat?ggtgcatttg?gtgaacaagg 2580
agtcgtcaga?aactccagac?cagtttatga?cagctgatga?gacaaggaac?ctgcagaatg 2640
tggacatgaa?gattggggtg?taacacctac?accattatct?tggaaagaaa?caaccgttgg 2700
aaacataacc?attacaggga?gctgggacac?ttaacagatg?caatgtgcta?ctgattgttt 2760
cattgcgaat?cttttttagc?ataaaatttt?ctactctttt?tgttttttgt?gttttgttct 2820
ttaaagtcag?gtccaatttg?taaaaacagc?attgctttct?gaaattaggg?cccaattaat 2880
aatcagcaag?aatttgatcg?ttccagttcc?cacttggagg?cctttcatcc?ctcgggtgtg 2940
ctatggatgg?cttctaacaa?aaactacaca?tatgtattcc?tgatcgccaa?cctttccccc 3000
accagctaag?gacatttccc?agggttaata?gggcctggtc?cctgggagga?aatttgaatg 3060
ggtccatttt?gcccttccat?agcctaatcc?ctgggcattg?ctttccactg?aggttggggg 3120
ttggggtgta?ctagttacac?atcttcaaca?gaccccctct?agaaattttt?cagatgcttc 3180
tgggagacac?ccaaagggtg?aagctattta?tctgtagtaa?actatttatc?tgtgtttttg 3240
aaatattaaa?ccctggatca?gtcctttgat?cagtataatt?ttttaaagtt?actttgtcag 3300
aggcacaaaa?gggtttaaac?tgattcataa?taaatatctg?tacttcttcg?atcttcacct 3360
tttgtgctgt?gattcttcag?tttctaaacc?agcactgtct?gggtccctac?aatgtatcag 3420
gaagagctga?gaatggtaag?gagactcttc?taagtcttca?tctcagagac?cctgagttcc 3480
cactcagacc?cactcagcca?aatctcatgg?aagaccaagg?agggcagcac?tgtttttgtt 3540
ttttgttttt?tgtttttttt?ttttgacact?gtccaaaggt?tttccatcct?gtcctggaat 3600
cagagttgga?agctgaggag?cttcagcctc?ttttatggtt?taatggccac?ctgttctctc 3660
ctgtgaaagg?ctttgcaaag?tcacattaag?tttgcatgac?ctgttatccc?tggggcccta 3720
tttcatagag?gctggcccta?ttagtgattt?ccaaaaacaa?tatggaagtg?ccttttgatg 3780
tcttacaata?agagaagaag?ccaatggaaa?tgaaagagat?tggcaaaggg?gaaggatgat 3840
gccatgtaga?tcctgtttga?catttttatg?gctgtatttg?taaacttaaa?cacaccagtg 3900
tctgttcttg?atgcagttgc?tatttaggat?gagttaagtg?cctggggagt?ccctcaaaag 3960
gttaaaggga?ttcccatcat?tggaatctta?tcaccagata?ggcaagttta?tgaccaaaca 4020
agagagtact?ggctttatcc?tctaacctca?tattttctcc?cacttggcaa?gtcctttgtg 4080
gcatttattc?atcagtcagg?gtgtccgatt?ggtcctagaa?cttccaaagg?ctgcttgtca 4140
tagaagccat?tgcatctata?aagcaacggc?tcctgttaaa?tggtatctcc?tttctgaggc 4200
tcctactaaa?agtcatttgt?tacctaaact?tatgtgctta?acaggcaatg?cttctcagac 4260
cacaaagcag?aaagaagaag?aaaagctcct?gactaaatca?gggctgggct?tagacagagt 4320
tgatctgtag?aatatcttta?aaggagagat?gtcaactttc?tgcactattc?ccagcctctg 4380
ctcctccctg?tctaccctct?cccctccctc?tctccctcca?cttcacccca?caatcttgaa 4440
aaacttcctt?tctcttctgt?gaacatcatt?ggccagatcc?attttcagtg?gtctggattt 4500
ctttttattt?tcttttcaac?ttgaaagaaa?ctggacatta?ggccactatg?tgttgttact 4560
gccactagtg?ttcaagtgcc?tcttgttttc?ccagagattt?cctgggtctg?ccagaggccc 4620
agacaggctc?actcaagctc?tttaactgaa?aagcaacaag?ccactccagg?acaaggttca 4680
aaatggttac?aacagcctct?acctgtcgcc?ccagggagaa?aggggtagtg?atacaagtct 4740
catagccaga?gatggttttc?cactccttct?agatattccc?aaaaagaggc?tgagacagga 4800
ggttattttc?aattttattt?tggaattaaa?tacttttttc?cctttattac?tgttgtagtc 4860
cctcacttgg?atatacctct?gttttcacga?tagaaataag?ggaggtctag?agcttctatt 4920
ccttggccat?tgtcaacgga?gagctggcca?agtcttcaca?aacccttgca?acattgcctg 4980
aagtttatgg?aataagatgt?attctcactc?ccttgatctc?aagggcgtaa?ctctggaagc 5040
acagcttgac?tacacgtcat?ttttaccaat?gattttcagg?tgacctgggc?taagtcattt 5100
aaactgggtc?tttataaaag?taaaaggcca?acatttaatt?attttgcaaa?gcaacctaag 5160
agctaaagat?gtaatttttc?ttgcaattgt?aaatcttttg?tgtctcctga?agacttccct 5220
taaaattagc?tctgagtgaa?aaatcaaaag?agacaaaaga?catcttcgaa?tccatatttc 5280
aagcctggta?gaattggctt?ttctagcaga?acctttccaa?aagttttata?ttgagattca 5340
taacaacacc?aagaattgat?tttgtagcca?acattcattc?aatactgtta?tatcagagga 5400
gtaggagaga?ggaaacattt?gacttatctg?gaaaagcaaa?atgtacttaa?gaataagaat 5460
aacatggtcc?attcaccttt?atgttataga?tatgtctttg?tgtaaatcat?ttgttttgag 5520
ttttcaaaga?atagcccatt?gttcattctt?gtgctgtaca?atgaccactg?ttattgttac 5580
tttgactttt?cagagcacac?ccttcctctg?gtttttgtat?atttattgat?ggatcaataa 5640
taatgaggaa?agcatgatat?gtatattgct?gagttgaaag?cacttattgg?aaaatattaa 5700
aaggctaaca?ttaaaagact?aaaggaaaca?gaaaaaaaaa?aaaaaaaa 5748
Claims (10)
1. the application of the hybridization in situ detection kit of a CD44 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 CD44 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 CD44 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910056178 CN101993941A (en) | 2009-08-10 | 2009-08-10 | In situ hybridization detection kit of CD44 gene and detection method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910056178 CN101993941A (en) | 2009-08-10 | 2009-08-10 | In situ hybridization detection kit of CD44 gene and detection method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101993941A true CN101993941A (en) | 2011-03-30 |
Family
ID=43784690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910056178 Pending CN101993941A (en) | 2009-08-10 | 2009-08-10 | In situ hybridization detection kit of CD44 gene and detection method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101993941A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103674913A (en) * | 2013-12-04 | 2014-03-26 | 南京邮电大学 | Fluorescence method and kit for testing lymphocyte homing receptor |
| WO2014144804A1 (en) * | 2013-03-15 | 2014-09-18 | Varian Medical Systems, Inc. | Biomarkers for radiation treatment |
| US10646730B2 (en) | 2017-02-24 | 2020-05-12 | Varian Medical Systems International Ag | Radiation treatment planning and delivery using collision free regions |
-
2009
- 2009-08-10 CN CN 200910056178 patent/CN101993941A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014144804A1 (en) * | 2013-03-15 | 2014-09-18 | Varian Medical Systems, Inc. | Biomarkers for radiation treatment |
| US9938583B2 (en) | 2013-03-15 | 2018-04-10 | Varian Medical Systems, Inc. | Biomarkers for radiation treatment |
| US10844437B2 (en) | 2013-03-15 | 2020-11-24 | Varian Medical Systems, Inc. | Biomarkers for radiation treatment |
| CN103674913A (en) * | 2013-12-04 | 2014-03-26 | 南京邮电大学 | Fluorescence method and kit for testing lymphocyte homing receptor |
| CN103674913B (en) * | 2013-12-04 | 2015-10-21 | 南京邮电大学 | A kind of fluorescent method and kit thereof detecting lymphocyte homing receptor |
| US10646730B2 (en) | 2017-02-24 | 2020-05-12 | Varian Medical Systems International Ag | Radiation treatment planning and delivery using collision free regions |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101993941A (en) | In situ hybridization detection kit of CD44 gene and detection method and application thereof | |
| CN101993913A (en) | In situ hybridization detection kit and detection method for ICAM1 gene and application | |
| CN101988095A (en) | In-situ hybridization detection kit for SCCA1 genes and detection method and application thereof | |
| CN101363046A (en) | Kit for broad spectrum cancer hybridization in situ, detection method and application thereof | |
| CN101988087A (en) | In-situ hybridization detection kit of p16 gene as well as detection method and application thereof | |
| CN101993935A (en) | In-situ hybridization assay kit and detection method for MTDH gene and application of kit | |
| CN101988089A (en) | In-situ hybridization detection kit and detection method of squamous cell carcinoma associated antigen (SCC) gene and application of detection kit | |
| CN101993940A (en) | In-situ hybridization detection kit for CHIP genes and detection method and application thereof | |
| CN101363050B (en) | Kit for CD326 gene hybridization in situ, detection method and application thereof | |
| CN101429548A (en) | DLK1 gene nucleic acid hybridization in situ detection kit, detection method and uses thereof | |
| CN101429542A (en) | Hybridization in situ detection kit for VMP1 gene, detection method and uses thereof | |
| CN101993920A (en) | In-situ hybridization detection kit for Survivin genes and detection method and application thereof | |
| CN101988101A (en) | In-situ hybridization detection kit of MK gene as well as detection method and application thereof | |
| CN101469349A (en) | In situ hybridization detection kit for lung cancer , detecting method and use thereof | |
| CN101851668A (en) | In situ hybridization detection kit of AP-1 gene, and detection method and application thereof | |
| CN101993937A (en) | Maspin gene in-situ hybridization detection kit and detection method and use thereof | |
| CN101429554B (en) | Detection kit for HCCR gene nucleic acid hybridization in situ, detection method and uses thereof | |
| CN101988113A (en) | In-situ hybridization detection kit for DCC genes and detection method and application thereof | |
| CN101993917A (en) | Cdc2 gene in-situ hybridization detection kit and detection method and use thereof | |
| CN101993947A (en) | In-situ hybridization detection kit for Mena genes and detection method and application thereof | |
| CN101993934A (en) | In situ hybridization detection kit for EZH2 gene, detection method and application thereof | |
| CN101993919A (en) | In-situ hybridization detection kit for PD-ECGF genes and detection method and application thereof | |
| CN101993942A (en) | In situ hybridization detection kit for cancer antigen 15-3 (CA15-3) genes, detection method and application thereof | |
| CN101469352A (en) | In situ hybridization detection kit for early uterocarcinoma, detecting method and use | |
| CN101993943A (en) | Kit for detecting in situ hybridization of romodomain-4 (BRD-4) genes, and detection method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SUZHOU FUYING GENE TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: RUIQU BIOTECHNOLOGY (SHANGHAI) CO., LTD. Effective date: 20110429 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 201108 ROOM 426, BUILDING 4, NO. 728, GUANGHUA ROAD, MINHANG DISTRICT, SHANGHAI TO: 215132 ROOM 307, BUILDING A2, BIOBAY, NO. 218, XINGHU STREET, SUZHOU INDUSTRIAL PARK |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20110429 Address after: 215132 Suzhou Industrial Park BioBAY Xinghu Street No. 218 building A2 room 307 Applicant after: Suzhou Fuying Gene Technology Co.,Ltd. Address before: 201108 room 4, building 728, Guanghua Road, 426, Shanghai, Minhang District Applicant before: Ruiqu Biotechnology (Shanghai) Co., Ltd. |
|
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20110330 |