[summary of the invention]
The objective of the invention is provides a kind of purposes of hybridization in situ detection kit of KiSS1 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 KiSS1 gene is provided.
Another purpose of the present invention is that a kind of in situ hybridization detection method of KiSS1 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 KiSS1 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 melanoma or mammary cancer.
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 KiSS 1 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 KiSS1 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 KiSS1 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.
[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 KiSS1 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 KiSS1 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 at present commonly used, and this method is by detecting the KiSS1 gene expression amount in the substrate cell, is used for determining whether cancer takes place and/or shift.
Clinical study shows that the KiSS1 gene has broad spectrum, because KiSS1 gene high expression level in the normal people.If KiSS1 genetic expression is low or zero expression, illustrates that cancer recurs, shifts, spreads, thereby obtain the diagnostic message of cancer.When detecting the KiSS1 expression of gene and be lower than normal control, to be that cancer is early stage shift or the cancer metastasis susceptible person then measurable experimenter, and these cancers comprise melanoma or mammary cancer.
The embodiment of the invention is sampled as: melanoma shifts 5 of patients, 5 of Metastasis in Breast 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 KiSS1 genes are not expressed the cell dye-free; Normal control group KiSS1 gene has overexpression, cell dyeing.Concrete outcome is asked for an interview Fig. 1, Fig. 2 and Fig. 3.
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.
<110〉Rui bends biotechnology (Shanghai) Co., Ltd.
<120〉a kind of hybridization in situ detection kit of KiSS1 gene and detection method thereof and application