CN102994630A - Ovarian clear cell carcinoma diagnostic kit and preparation method thereof - Google Patents
Ovarian clear cell carcinoma diagnostic kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a fluorescence in situ hybridization detection kit prepared by utilizing an HNF1B fluorescent probe, and also relates to a preparation method of the HNF1B fluorescent probe. The HNF1B fluorescent probe and the corresponding kit can be used for diagnosis on ovarian clear cell carcinoma and selection of treatment plan.
Description
Technical field
The present invention relates to a kind of fluorescence in situ hybridization detection test kit of the HNF1B of utilization fluorescent probe preparation, the preparation method who also relates to the HNF1B fluorescent probe, HNF1B fluorescent probe of the present invention and corresponding test kit can be used for the clear cell carcinoma of ovary diagnosis and treatment plan is selected.
Background technology
Ovarian cancer (ovarian cancer) is one of common tumour of female sex organ, and sickness rate is only second to cervical cancer and carcinoma of uterine body and is listed as and occupies the 3rd.But because ovarian cancer causes the dead, but account for the first place of all kinds of gynecological tumors, women's life is caused serious threat.The cause of disease of ovarian cancer it be unclear that, and its morbidity may be relevant with age, fertility, blood group, mental element and environment etc.Ovarian cancer is clinical asymptomatic in early days, differentiates its types of organization and good pernicious difficult, so far, and clinical data statistics five year survival rate only 25%～30%.Ovarian cancer is relatively common disease, and nearly 1.4% women can suffer from this disease.Find that early 90% patient can both survive; Find that cancer cells is diffused into ovary late, survival rate is lower than 30%.Clear cell carcinoma of ovary (Ovarian clear cell carcinoma, OCCA) account for 5%～11% of ovarian cancer, 58 years old mean age of morbidity, in close relations with endometriosis, merging endometriosis in the ovary of pathology reaches about 24%, and having simultaneously the reaching about 25%～50% of endometriosis of pelvis pathology, this is rare in other epithelial ovarian cancers.Clear cell carcinoma of ovary is malignant tumour, and prognosis is poorer than other somatotype, with clinical stages substantial connection is arranged.
The ovarian cancer inspection has several method, comprises cytodiagnosis, ultrasound investigation, immunologic test etc., but all very not successful.At present, the susceptibility of malignant tumor of ovary mark and specificity all can not satisfy the needs of early diagnosis, multiplexly detect in the treatment and/or the change of illness state after the treatment, for the evaluation curative effect with find that in time tumor recurrence provides foundation.
Hepatocyte neclear factor-1 (HNF1 homeobox B, HNF1B) be positioned at long-armed 1 district of karyomit(e) 2 bands No. 17, a kind of transcription factor superfamily member that comprises homeodomain of encoding, proteins encoded is combined with DNA and is formed homodimer, or forms heterodimer with HNF1a (hepatocyte nuclear factor 1-alpha) combination.Transcription factor HNF1B forms and keeps glycaemic homeostasis pancreatic cell has vital role.The transgenation meeting causes renal cyst and non insulin dependent diabetes or is called the Second-Type diabetes, and the expression of this gene can change in certain cancers.There are some researches show that at present HNF1B is the biomarker of clear cell carcinoma of ovary, can effectively distinguish clear cell carcinoma and other pathology type.Disturb this genetic expression by RNA in the clinical study, can reach the apoptosis purpose, this gene is had broad application prospects aspect treatment.[Jin?L，et?al.Regulation?of?tissue-specific?expression?of?renal?organic?anion?transporters?by?hepatocyte?nuclear?factor?1α/βand?DNA?methylation.J?Pharmacol?Exp?Ther，2012Mar；Stanislas?Faguer，et?al.Diagnosis，management，and?prognosis?ofHNF1B?nephropathy?in?adulthood.Kidney?International80，768-776；?Berndt?SI，Sampson?J，Yeager?M，et?al.Large-scale?fine?mapping?of?the?HNF1B?locus?and?prostate?cancer?risk.Hum?Mol?Genet.2011Aug?15；20(16)：3322-9；Brimo?F，Herawi?M，Sharma?R，et?al.Hepatocyte?nuclear?factor-1βexpression?in?clear?cell?adenocarcinomas?of?the?bladder?and?urethra：diagnostic?utility?and?implications?for?histogenesis.Kim?HJ，Bae?JS，Lee?J，et.al.HNF1Bpolymorphism?associated?with?development?of?prostate?cancer?in?Korean?patients.Hum?Pathol.2011Nov；42(11)：1613-9.Urology.2011Oct；78(4)：969.e1-6；Sohei?Yamamoto，Hitoshi?Tsuda，Shinsuke?Aida，et，al.Immunohistochemical?detection?of?hepatocyte?nuclearfactor?1βin?ovarian?and?endometrial?clear-cell?adenocarcinomas?and?nonneoplastic?endometrium.Human?Pathology?Vol.38.Issue?7，1074-1080；K.Yamaguchi，M.Mandai，T.Oura，N.Matsumura，J.Hamanishi?and?T.Baba?et?al.，Identification?of?an?ovarian?clear?cell?carcinoma?gene?signature?that?reflects?inherent?disease?biology?and?the?carcinogenic?processes，Oncogene?29(2010)，pp.1741-1752.]
At present testing laboratory's detection method common are based on the expression analysis of immunohistochemical methods with based on sudden change and the deletion analysis of fluorescence quantifying PCR method (FQ-PCR) or sequencing technologies.[Harries?LW，Brown?JE，Gloyn?AL(2009)Species-Specific?Differences?in?the?Expression?of?the?HNF1A，HNF1Band?HNF4AGenes.PLoSONE?4(11)：e7855；Immunohistochemical?detection?of?hepatocyte?nuclearfactor?1βin?ovarian?and?endometrial?clear-cell?adenocarcinomas?and?nonneoplastic?endometrium.Human?Pathology?Vol.38，Issue?7，1074-1080]
To sum up, HNF1B is expected to differentiate as tumour the new target drone of somatotype and targeted therapy because of its specifically expressing feature in clear cell carcinoma of ovary.But the detection method of this gene only rests on testing laboratory's stage at present, the detection reagent that the market non sensitivity is high, specificity is good.
Fluorescence in situ hybridization technique with the complementary hybridization of the DNA/RNA to be measured on known array labeled DNA probe and the slide glass, detects the hybridization signal judged result based on the base complementrity principle under fluorescent microscope.The FISH technology connects cytogenetics and molecular biology change, allows small gene alteration be revealed under the naked eyes, has expanded the scope that cytogenetics detects, and has significantly improved the ability of its identification abnormal chromosome.The detections such as gene amplification, translocation rearrangement and disappearance in the tumor research have been widely used at present.
The present invention utilizes fluorescence in situ hybridization technique, take the HNF1B constant gene segment C as target, and design and preparation fluorescent probe.Utilize the probe realization to the detection of HNF1B gene appearance, help clear cell carcinoma of ovary to differentiate somatotype and treatment plan formulation, early differentiate early treatment thereby reach, the raising treatment effect.
The invention provides a kind of preparation method of clear cell carcinoma of ovary molecular marker HNF1B probe, and the HNF1B gene detecting kit that utilized on this basis the probe independent development, filled up the blank of domestic this detection field, have very great meaning.
Summary of the invention
An object of the present invention is to provide HNF1B gene probe (GSP HNF1B) and as the preparation method who is used for No. 17 karyomit(e) centromeric probes (CSP17) that No. 17 karyomit(e) differentiates of internal control.
Preferred embodiment according to the present invention, the preparation process of GSP HNF1B comprises:
(1) colony screening: all contain HNF1B (17q12) gene cloning by NCBI Mapview database retrieval, and these clones are screened, and select optimum clone.
(2) clone's culture ﹠ identification: the clone who determines according to screening numbers the corresponding clone of purchase, gets 100 μ l clone bacterium liquid and is added in the TB nutrient solution (chlorampenicol resistant) of 500ml, shakes bacterium in 37 ℃ of shaking tables and cultivates 8～12 hours; Use corresponding STS primer to carry out pcr amplification for the HNF1B gene probe, and by 2% agarose gel electrophoresis the amplified production fragment is analyzed, thereby finish clone's to be selected evaluation.
(3) gene probe preparation: to identifying positive bacterium liquid, carry out the extraction of plasmid DNA; Mensuration by the OD value is carried out quantitatively plasmid DNA; Then, plasmid DNA is carried out fluorescent mark; Marked product is carried out purifying; Obtain probe, lucifuge ,-20 ± 5 ℃ of storages.
(4) gene probe checking: use human proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.
Preferred embodiment according to the present invention, the preparation process of CSP17 comprises:
(1) fragment analysis: by NCBI Mapview database retrieval repeat region sequence, carry out repeatability analysis.
(2) design of primers: for repeated fragment, utilize Primer Premier 5.0 design primers pair.
(3) clone, culture ﹠ identification: take the human gene group DNA as template, utilize above-mentioned primer to carrying out the PCR reaction.Identify by electrophoresis; The purpose product is cloned, cultivated, use equally above-mentioned primer to carrying out pcr amplification, and by 2% agarose gel electrophoresis the amplified production fragment is analyzed, thereby finish clone's to be selected evaluation.
(4) probe preparation: to identifying positive bacterium liquid, carry out the extraction of plasmid DNA; Mensuration by the OD value is carried out quantitatively plasmid DNA; Then, plasmid DNA is carried out fluorescent mark; Marked product is carried out purifying; Obtain probe, lucifuge ,-20 ± 5 ℃ of storages.
(5) probe checking: use human proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.
Preferred embodiment according to the present invention contains the most preferred clone of HNF1B gene in the colony screening step, be numbered CTD-2257N17 (chr17:36033624..36152533).
Preferred embodiment according to the present invention, the STS primer that uses in clone's culture ﹠ identification step to sequence is: upstream primer 5 '-TGCACAGACTACAACCCTAAACC-3 ' (SEQ ID NO:1) and downstream primer 5 '-TGTAGCACTGTTGTTCTTTGGG-3 ' (SEQ ID NO:2); The pcr amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.
Preferred embodiment according to the present invention for the preparation of the primer of CSP17 probe to sequence is: upstream primer 5 '-TTGTAGAATCTGCGAAGGGAC-3 ' (SEQ ID NO:3) and downstream primer 5 '-AGTGTTTCCAAACTGCTGAATC-3 ' (SEQ ID NO:4); The pcr amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.
Preferred embodiment according to the present invention, the cloned plasmids DNA extraction can be used commercially available plasmid extraction kit in the probe preparation process, the Plasmid Maxi Kit of preferred Qiagen company.
Preferred embodiment according to the present invention, cloned plasmids DNA's quantitatively is the absorbancy that will measure respectively after the plasmid DNA dilution under 260nm and the 280nm in the probe preparation process, calculates production concentration.
Preferred embodiment according to the present invention, plasmid DNA is carried out fluorescent mark and can be used nick translation method mark in the probe preparation process, utilizes DNaseI and dna polymerase i to realize the fluorescent mark of gene probe.
Preferred embodiment according to the present invention, in the 50 μ l nick translation systems dna polymerase i usage quantity between 10U～20U, DNase I usage quantity between 0.001U～0.01U, flag condition be 16 ℃ 2 hours.
Preferred embodiment according to the present invention, the fluorescein of probe mark is fluorescein-labeled dUTP, and preferred Spectrum dUTP.
Preferred embodiment according to the present invention, probe concentration method are that the ethanol precipitation is concentrated, use at last 1～2 μ l sterilization purified water or Human Cot-1DNA (1 μ g/ μ l) dissolution precipitation.
Another object of the present invention is to utilize HNF1B gene probe and a kind of HNF1B gene by fluorescence hybridization in situ detection kit for assisting clear cell carcinoma of ovary discriminating somatotype and treatment to select of CSP17 probe (internal control) preparation.
In order to realize the present invention, we have adopted following technical scheme:
(1) sample process and film-making: sample is processed according to in-situ hybridization method.
(2) hybridization: preparing hybrid liquid mainly comprises label probe and hybridization buffer.Carry out 8～16 hours hybridization under the suitable temp, then with suitable washing lotion flush away not in conjunction with upper and probe non-specific binding; The DAPI counterstain is redyed.
(3) observe fluorescent signal by the corresponding filter block of fluorescent microscope, unlike signal is observed counting.
Test kit based on above technical scheme invention comprises: 1) hybridization buffer, fluorescence labeling probe mixture, unlabelled competitive DNA, and the DAPI counterstain, and 2) separate and the concentrated packing box of packing these reagent bottles or pipe.
According to a preferred embodiment of the invention, the component of hybridization buffer comprises deionized formamide, SSC, and T 500, wherein deionized formamide concentration is 50%～70%, T 500 concentration is 0.1g/ml.
According to a preferred embodiment of the invention, the fluorescence labeling probe mixture comprises GSP HNF1B and CSP17 probe, and the usage quantity of two kinds of gene probes is 0.5 μ l in every person-portion fluorescence labeling probe mixture.
According to a preferred embodiment of the invention, by hybridization buffer, fluorescence labeling probe mixture and unlabelled competitive DNA preparing hybrid liquid, wherein unlabelled competitive DNA selects Human COT-1DNA.Add 7 μ l hybridization buffers in every person-portion hybridization solution, 1 μ l fluorescence labeling probe mixture, Human COT-1DNA usage quantity is 1 μ g, and mends to 10 μ l with H2O.
According to a preferred embodiment of the invention, wherein DAPI counterstain compound method is that 50～250ng DAPI is dissolved in the anti-liquid (10mg/ml Ursol D/PBS, glycerine mixed solution) that fades of 1ml.
Utilize test kit of the present invention, according to the fluorescence in-situ hybridization method of routine to mid-term of people and interval cell carry out signal-count.
The present invention compared with prior art has following advantages:
(1) realized detection to clear cell carcinoma of ovary recruit mark HNF1B gene appearance;
(2) carry out accurately and rapidly signal-count and as a result good reproducibility;
(3) need not to carry out cell cultures and the high-quality Metaphase Chromosome sheet of preparation, can be used for mid-term or interval cell signal counting, operate relatively simple;
(4) by being prepared into test kit, can be implemented in the application in the fields such as oncobiology, cytogenetics, help each molecular marker of comprehensive evaluation, understand contacting of itself and tumour generation etc.
Description of drawings
Fig. 1 shows the electrophoresis result of GSPHNF1B clone identification.Purpose product 223bps.Wherein 1 road is marker, and 2 roads are sample; Arrow indicates and represents 300bp.
Fig. 2 shows the electrophoresis result of CSP17 clone identification.Purpose product 316bps.Wherein 1 road is marker, and 2 roads are sample; Arrow indicates and represents 300bp.
Fig. 3 shows GSP HNF1B (redness) and CSP17 (green) detected result on human peripheral blood lymphocyte's Metaphase Chromosome.
Fig. 4 shows the detected result of GSP HNF1B (redness) and CSP17 (green) in the clear cell carcinoma of ovary tissue samples.Wherein arrow indicates respectively the fluorescent signal of dichromatism probe.
Fig. 5 shows the detected result of GSP HNF1B (redness) and CSP17 (green) in the ovarian serous cystocarcinoma tissue samples.Wherein arrow indicates respectively the fluorescent signal of dichromatism probe.
Fig. 6 shows HNF1B gene place 17q12 section.
Embodiment
The following example is intended to illustrate rather than limit the present invention.
The preparation of embodiment 1:GSP HNF1B probe
(1) design of primers colony screening: the HNF1B gene is positioned at human chromosome 17q12 section, and all contain the HNF1B gene cloning NCBI Mapview database retrieval, filters out to include this gene cloning, is numbered CTD-2257N17.(referring to accompanying drawing 6)
(2) clone's culture ﹠ identification: buy clone CTD-2257N17, get 100 μ l clone bacterium liquid and be added in the TB nutrient solution (chlorampenicol resistant) of 500ml, shake bacterium in 37 ℃ of shaking tables and cultivated 8～12 hours; Bacterium liquid uses upstream primer 5 '-TGCACAGACTACAACCCTAAACC-3 ' and downstream primer 5 '-TGTAGCACTGTTGTTCTTTGGG-3 ' to carry out pcr amplification, and amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production is carried out the electrophoresis checking, and the result has bright band (referring to accompanying drawing 1) at 223bps.
(3) gene probe preparation: identify positive bacterium liquid, use the Plasmid Maxi Kit of Qiagen company, the working method that requires is to specifications carried out extraction of plasmid DNA, and is quantitative to plasmid DNA by the absorbancy of measuring 260nm and 280nm place; According to formula: double-stranded DNA concentration (ng/ μ l)=OD260 * 50 (ng/ μ l) calculates plasmid DNA concentration.
By the nick translation method plasmid DNA is carried out fluorescent mark, the fluorescein of probe mark is Spectrum dUTP, selects orange-dUTP mark HNF1B gene.By following scheme, prepare the PCR reaction system under the strict lucifuge condition on ice.
Joined rear concussion mixing, 16 ℃ of marks 2 hours are hatched 10 minutes inactivators for 80 ℃ again.
Marked product is carried out ethanol precipitation and concentrated, in the 1.5ml centrifuge tube, adds successively sodium-acetate and dehydrated alcohol by following scheme, lucifuge, preparation on ice:
Mixing is placed in-80 ℃ of refrigerators 2 hours, and centrifugal 20 minutes of 4 ℃ of 12000rpm carefully remove supernatant, stir precipitation, add 70% ethanol of 1ml, 4 ℃ 12000 rev/mins centrifugal 10 minutes, carefully remove supernatant, stir precipitation, lucifuge is dry.Use 1 μ l sterilization purified water dissolution precipitation, obtain the HNF1B gene probe, lucifuge ,-20 ℃ of storages.
(4) HNF1B gene probe checking: use human proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.Comprise mid-term or interphase chromosome DNA, behind the fluorescence in situ hybridization, interval cell or the upper equal visible fluorescence signal of Metaphase Chromosome (No. 10 karyomit(e)).
The preparation of embodiment 2:CSP17 centromeric probe
(1) design of primers: by NCBI Mapview database retrieval repeat region sequence, carry out repeatability and analyze, utilize Primer Premier 5.0 design primers pair.
(2) clone, culture ﹠ identification: take the human gene group DNA as template, utilize upstream primer 5 '-TTGTAGAATCTGCGAAGGGAC-3 ' (SEQ ID NO:3) and downstream primer 5 '-AGTGTTTCCAAACTGCTGAATC-3 to carry out the PCR reaction.Amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production is carried out the electrophoresis checking, and the result has bright band (referring to accompanying drawing 2) at 316bps.
(3) probe preparation: identify positive bacterium liquid, use the Plasmid Maxi Kit of Qiagen company, the working method that requires is to specifications carried out extraction of plasmid DNA, and is quantitative to plasmid DNA by the absorbancy of measuring 260nm and 280nm place; According to formula: double-stranded DNA concentration (ng/ μ l)=OD260 * 50 (ng/ μ l) calculates plasmid DNA concentration.
By the nick translation method plasmid DNA is carried out fluorescent mark, the fluorescein of probe mark is Spectrum dUTP, selects green-dUTP mark CSP17 probe.By following scheme, prepare the PCR reaction system under the strict lucifuge condition on ice.
Joined rear concussion mixing, 16 ℃ of marks 2 hours are hatched 10 minutes inactivators for 80 ℃ again.
Marked product is carried out ethanol precipitation and concentrated, in the 1.5ml centrifuge tube, adds successively sodium-acetate and dehydrated alcohol by following scheme, lucifuge, preparation on ice:
Mixing is placed in-80 ℃ of refrigerators 2 hours, and centrifugal 20 minutes of 4 ℃ of 12000rpm carefully remove supernatant, stir precipitation, add 70% ethanol of 1ml, 4 ℃ 12000 rev/mins centrifugal 10 minutes, carefully remove supernatant, stir precipitation, lucifuge is dry.Use 1 μ l sterilization purified water dissolution precipitation, obtain the HNF1B gene probe, lucifuge ,-20 ℃ of storages.
(4) CSP17 probe checking: use human proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.Comprise mid-term or interphase chromosome DNA, behind the fluorescence in situ hybridization, interval cell or the upper equal visible fluorescence signal of Metaphase Chromosome (No. 17 karyomit(e)).
The preparation of embodiment 3:HNF1B gene by fluorescence hybridization in situ detection kit
Take 10 person-portions/box as example.
(1) hybridization solution preparation
The probe that mark is good is put well successively, according to the form below scheme preparation fluorescence labeling probe mixture:
According to the form below scheme preparing hybrid liquid:
(2) DAPI counterstain preparation
The anti-liquid that fades: the necessary lucifuge of substantial length of operation, the Ursol D of 10mg is dissolved among the PBS of 1ml, and regulating pH is 9.0, adds 9ml glycerine, repeatedly shakes mixing ,-20 ℃ of storages.Whole solution should be for colourless or slightly faint yellow, if present yellow or the orange abandoned well that then needs is prepared again.
With deionized water preparation 1mg/ml DAPI storage liquid.
The DAPI solution (0.1mg/ml) of getting 2.5 μ l is dissolved in the anti-liquid that fades of 1ml, repeatedly shakes mixing under the lucifuge condition ,-20 ℃ of airtight preservations of lucifuge.
(3) finished product assembling
| The component title | Specification | Quantity |
| Hybridization solution | 100 μ l/ |
1 pipe |
| The DAPI counterstain | 100 μ l/ |
1 pipe |
| Specification sheets | ? | 1 part |
The using method of embodiment 4:HNF1B gene by fluorescence hybridization in situ detection kit in the fixing paraffin-embedded ovary tissue sample of formalin
(1) slide pre-treatment
Slide is put into the roasting sheet of 65+5 ℃ of thermostat container and is spent the night; Take out slide, put into room temperature dimethylbenzene 30 minutes, put it into again room temperature 1: 1 (V: dimethylbenzene V): in the dehydrated alcohol 10 minutes, put into the room temperature dehydrated alcohol 10 minutes, put into successively again room temperature 100% ethanol, 90% ethanol, 70% ethanol each 3 minutes; Left standstill in the deionized water at room temperature 3 minutes, and drew excessive moisture; Boil sheet in 100 ± 5 ℃ the deionized water 20 minutes (section is placed horizontally in the container, and sample faces up).Stomach en-reaction solution at sample areas dropping 200ul digested 15 minutes.Get rid of unnecessary liquid, put into 2 * SSC room temperature washing 5 minutes; Take out, put into again another cylinder room temperature 2 * SSC 5 minutes; Take out, put into again 70%, 90%, 100% gradient ethanol dehydration each 2 minutes; Take out slide, room temperature is dried slide; Continue crossover process.
(2) the same time variation of sample and probe
From test kit, take out hybridization solution, the concussion mixing, instantaneous centrifugal; Add the hybridization solution of 10 μ l to the hybridization zone, covered gently presses hybridization solution is evenly distributed rapidly, avoids producing bubble; Rubber cement covers the edge that cover glass contacts with slide glass fully along cover glass edge mounting; Slide is put into hybridization instrument, and moistening in situ hybridization instrument humidity bar inserts wet bar, covers the hybridization instrument loam cake, and " Denat﹠amp is set; Hyb " program, 85 ℃ of sex change 2 minutes, hybridize 37 ℃ 10～18 hours.Continue the post-hybridization washing step.
(3) post-hybridization washing and redying
Take out, carefully remove rubber cement and cover glass; Slide was put into 37 ± 1 ℃ of 2 * SSC 10 minutes; Putting it into 37+1 ℃ of 0.1%NP-40/2 * SSC washed 2 minutes again; Room temperature 70% ethanol 3 minutes; Dry the dark place.
Redye: drip 10 μ l DAPI to the slide glass target area, covered, the light pressure avoids producing bubble, in the dark deposits, and be to be seen.
(4) interpretation of result
Under Olympus BX53 fluorescent microscope, observe respectively the hybridization fluorescent signal that DAPI redyes with the filter group, use the CCD photographic recording.Under 40 * object lens, seek, under 100 * object lens, count; Adjust suitable focal length, signal and background are had clear and definite concept; Signaling point is because being positioned at cell; When there is fluorescent signal point in the extracellular, note with cell in signaling point distinguish, preferably can avoid this zone and count; Adjusting focal length finds signaling point in the different levels of examining; The number of each probe in each cell observed in record.
(5) result judges
Operate record GSP HNF1B and CSP 17 signal numbers by the treatment process requirement.The fluorescence in situ hybridization result shows: in human peripheral lymphocyte, the corresponding target spot on the Metaphase Chromosome can be seen respectively two signaling points (referring to accompanying drawing 3), and other chromosomal region has no fluorescent signal.In tissue samples, visible double-colored fluorescence probe signal in the nuclear, positive sample is seen 3R2G signal (referring to accompanying drawing 4), negative sample is seen 2R2G signal (referring to accompanying drawing 5).
The clinical in-service evaluation of embodiment 5:HNF1B gene detecting kit
Take clinical detection as clear cell carcinoma of ovary, Endometrium sample cancer, ovarian serous and mucous cystoadenocarcinoma be as detected object.Utilize the DDIT3 gene detecting kit described in the embodiment of the invention 3 to detect.Detected result such as table 1 show.
Table 1 detected result
As can be known from the results, 2 routine pernicious clear cell carcinoma detected results are the FISH positive, and other 7 routine sample standard deviation is negative.The HNF1B detection is used in prompting in clinical detection can assist clinical diagnosis.
Above content is the further description of the present invention being done in conjunction with concrete preferred implementation, can not assert that implementation of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. the HNF1B gene appearance fluorescence in situ hybridization detection test kit of an auxiliary clear cell carcinoma of ovary diagnosis and treatment plan selection, comprise: 1) hybridization buffer, fluorescence labeling probe mixture, unlabelled competitive DNA, DAPI counterstain, with 2) separate and the concentrated packing box of packing these reagent bottles or pipe, it is characterized in that the fluorescence labeling probe mixture comprises GSPHNF1B probe and No. 17 karyomit(e) centromeric probes, the usage quantity of GSP HNF1B and CSP17 probe is 0.5 μ l in every person-portion fluorescence labeling probe mixture.
2. according to claim 1 test kit is further characterized in that unlabelled competitive DNA is Human COT-1DNA.
3. according to claim 1 test kit is further characterized in that the component of hybridization buffer comprises deionized formamide, SSC, and T 500, wherein deionized formamide concentration is 50%～70%, T 500 concentration is 0.1g/ml.
4. according to claim 1 test kit is further characterized in that DAPI counterstain compound method is that 50～250ng DAPI is dissolved in the anti-liquid (10mg/ml Ursol D/PBS, glycerine mixed solution) that fades of 1ml.
5. method for preparing the described HNF1B gene probe of claim 1 test kit and No. 17 karyomit(e) centromeric probes is as follows:
(1) fragment screening: all contain the HNF1B gene cloning by NCBI Mapview database retrieval, and these clones are screened, selection contains the clone of HNF1B gene optimum, is numbered CTD-2257N17 (chr17:36033624..36152533); By No. 17 karyomit(e) repeat regions of NCBI Mapview database retrieval sequence, and carry out repeatability analysis, select the design of primers zone;
(2) culture identification: the clone who determines according to the HNF1B screening numbers purchase clone (Invitrogen), cellar culture, use is carried out pcr amplification for the STS primer of HNF1B gene region, and by 2% agarose gel electrophoresis the amplified production fragment is analyzed, thereby finish HNF1B clone's to be selected evaluation; CSP17 utilizes the primer of design to carrying out the PCR reaction take the human gene group DNA as template, identifies by electrophoresis; The purpose product is cloned, cultivated, identify thereby finish CSP 17 fragments to be selected;
(3) probe preparation: to identifying positive bacterium liquid, carry out the extraction of plasmid DNA; Mensuration by the OD value is carried out quantitatively plasmid DNA; Then, by the nick translation method plasmid DNA is carried out fluorescent mark; Marked product is carried out purifying;
(4) probe checking: use human proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.
6. preparation method according to claim 5 is further characterized in that the STS primer that uses in the clone's culture ﹠ identification step that comprises the HNF1B gene fragment is respectively sequence: upstream primer 5 '-TGCACAGACTACAACCCTAAACC-3 ' and downstream primer 5 '-TGTAGCACTGTTGTTCTTTGGG-3 '; The pcr amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.
7. preparation method according to claim 5 is further characterized in that the primer of preparation CSP17 probe to sequence is:
Upstream primer 5 '-TTGTAGAATCTGCGAAGGGAC-3 ' and downstream primer 5 '-AGTGTTTCCAAACTGCTGAATC-3 '; The pcr amplification condition is: 94 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.
8. preparation method according to claim 5 is further characterized in that the dna polymerase i usage quantity is between 10U～20U in the 50 μ l nick translation systems in the probe preparation process, and DNase I usage quantity is between 0.001U～0.01U.
9. preparation method according to claim 5, the fluorescein that is further characterized in that probe mark is fluorescein-labelled Spectrum dUTP.
10. preparation method according to claim 5 is further characterized in that the probe concentration method is that the ethanol precipitation is concentrated, uses 1～2 μ l sterilization purified water or Human Cot-1DNA (1 μ g/ μ l) dissolution precipitation at last.
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| CN (1) | CN102994630A (en) |
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| CN103276060A (en) * | 2013-04-28 | 2013-09-04 | 中国医学科学院血液病医院（血液学研究所） | Gene probe composition and kit for detecting epithelial ovarian cancer |
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Application publication date: 20130327 |