CN103131757B - Ovarian cancer individualized treatment detection reagent case and application thereof - Google Patents

Abstract

The invention relates to an ovarian cancer individualized treatment detection reagent case and application of the ovarian cancer individualized treatment detection reagent case, in particular to the ovarian cancer individualized treatment. The ovarian cancer individualized treatment detection reagent case and the application of the ovarian cancer individualized treatment detection reagent case comprise development and progression, staging and grading, forecast treatment and preparation of related molecular marker detection probes. The related molecular marker detection probes can be used for guiding the planning of an ovarian cancer individualized treatment schedule, evaluating the treatment effect, monitoring recurrence and metastasis, estimating prognosis, and the like.

Description

A kind of detection kit of ovarian cancer individualized treatment and application thereof

Technical field

The present invention relates to a kind of ovarian cancer individualized treatment detection agent and the application in ovarian cancer detects thereof.Particularly, relate to ovarian cancer individualized treatment, comprise develop, the preparation of the related molecule sign detection probes such as stage and step, prediction curative effect.The ovarian cancer molecular marker detection probes related in the present invention can be used for instructing the solution formulation of ovarian cancer individualized treatment, evaluates result for the treatment of, monitoring recurrence and transfer, evaluate its prognosis etc.

Background technology

Ovarian cancer is the malignant tumour betiding ovary tissue, and sickness rate is only second to cervical cancer and carcinoma of uterine body, accounts for the 3rd of female malignant.Because ovarian tumor is positioned at pelvic cavity, at few reveal any symptoms of ill initial stage, incidence of occult, progress rapidly, have been late period when the ovarian cancer patients of 70% ~ 80% finds, within 5 years, survival rate is only 20% ~ 30%, and the survival rate of early ovarian cancer patient can reach 90%.At present, the clinical complex therapy generally carrying out to perform the operation as main, but because pathological classification is many, complex structure, biological characteristics difference is large, and different to the susceptibility of chemotherapy, radiotherapy etc., causes the difficulty in process and affects the treatment, its mortality ratio exceedes cervical cancer and carcinoma of uterine body sum, accounts for the first place of gynecological tumor.Therefore, be only improved the early diagnosis level of ovarian cancer, grasp the biological nature of all types of malignant tumor of ovary, explore effective composite treatment, favourable therapic opportunity can be striven for, improve prognosis, improve the survival rate of ovarian cancer, reduce mortality ratio.

The same with most of malignant tumour, the evolution of ovarian cancer and deterioration are also the complex processes of a multi-step, multifactor participation, may relate to the common adjustment of multiple gene.The tumor markers relevant to ovarian cancer, along with immunology, molecular biological development, have also been obtained and more and more further investigate, and plays an important role in ovarian cancer clinical diagnosis and treatment.A large amount of clinical comparison and retrospective research find have multiple gene relevant to ovarian cancer.As higher in the BRCA1 genovariation ratio that has ovarian cancer, the expression of BRCA1 and P53 in ovarian epithelial carcinoma is negative correlativing relation, synergy mutually may be there is in both in the generation of ovarian cancer, evolution, the differentiation of common promotion ovarian cancer and deterioration (Zhihui Feng, Lisa Kachnic, JunranZhang, et, al.DNA Damage Induces p53-dependent BRCA1 Nuclear Export.The Journal ofBiological Chemistry, July 2,2004.279.28574-28584.).RAD51C genetic flaw ratio in ovarian cancer is higher, ZNF361, PSMA2, AIB1 be high expression level in ovarian cancer, ZNF217 and ovarian cancer significant correlation, cause developing of ovarian cancer, WT1 gene high expression level in ovarian epithelial carcinoma, relevant to classification neoplasm staging, PTEN expression amount in ovarian cancer progression declines gradually, etc.

Targeted therapy is as a kind of new oncotherapy means, and can improve the lethality of drug on tumor cell, and reduce the undesirable action of normal tissue organ simultaneously, comparatively traditional treatment mode demonstrates clear superiority, is progressively applied to clinical treatment.Along with the develop rapidly of the basic subjects such as molecular biology, immunology, virusology and genetically engineered, and people's going deep into that tumor development is studied, safe with it as a kind of new Therapeutic mode, the efficient characteristic of targeted therapy, new field has been started in treatment for ovarian cancer, become another methods for the treatment of likely after operation, radiotherapy, chemotherapy, become the new trend of current treatment of ovarian cancer.And there is obvious individual difference in the research different treatment plan of display and curative effect of medication, utilize molecular Biological Detection assess patient, treatment plan is formulated according to molecule parting, carry out personalized medicines, for raising treatment effect, reduce mortality ratio, reduce medical treatment cost and all have meaning (Albertson, D.G., 2006.Gene amplification in cancer.TrendsGenet.22 (8), 447-455.Albertson, D.G., Collins, C., McCormick, F., Gray, J.W., 2003.Chromosome aberrations in solid tumors.Nat.Genet.34 (4): 369-376.Ena Segota MD, Ronald M, Bukowski MD:The promise of targeted therapy:Cancer drugs become more specific.Cleveland Clinic Journal OfMedicine 2004, volume71, number 7.).At present, the genetic marker relevant to ovarian cancer, generation development genes involved and the gene studies of medication outcome prediction are more, but mostly rest on the laboratory study stage, market lack corresponding molecular diagnosis agents, greatly limit the development of personalized medicines.

The present invention is according to clinical study results, select the molecular indexes relevant to ovarian cancer disease process and outcome prediction two aspect as detecting target spot, by the expression predicting tumors biological behaviour feature of joint-detection in ovarian cancer, and as the index of judging prognosis, chemotherapy regimen can be adjusted by the monitoring to these genes and expression product thereof clinically.Specifically comprise as follows:

1. BRCA1 (breast cancer susceptibility gene 1): be positioned human chromosomal 17q21 district.Breast cancer susceptibility gene, cancer suppressor gene, it is encoded cancer suppressor protein, participates in many important cell activities, as the cell cycle regulation and control, transcription activating and suppression, Chromatin Remodeling, centrosome copy, DNA damage reparation etc.Normal BRCA1 genes encoding BRCA1 albumen, plays restraining effect to the growth of tumour.If due to congenital heredity or the reason producer sudden change day after tomorrow, gene product event resolves or reduction, just may cause the generation of ovarian tumor.Research shows, BRCA1 is high expression level in normal ovarian tissue, express in ovarian cancer and reduce, and along with cancer pathology histological grading increase and lymphatic metastasis occur time, positive expression rate declines gradually (Joshua Z Press, Alessandro De Luca, Niki Boyd, et, al.Ovarian carcinomas with genetic andepigenetic BRCA1 loss have distinct molecular abnormalities.BMC Cancer.2008,8:17; Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, et, al.A strong candidate for the breast and ovarian cancersusceptibility gene BRCA1.1994,266 (5182): 66-71).

2. PTEN (Phosphatase and tensin homolog): be positioned human chromosomal 10q23 district.PTEN is the cancer suppressor gene that first of finding up to now has phosphatase activity, play a part complicated and important in the cell signal transmission of somatomedin, regulate the sticking of cell by T suppression cell mitotic division, cell death inducing, participation, shift and the effect of the performance cancer suppressor gene such as differentiation.The disappearance of PTEN and sudden change and kinds of tumors, the generation and the correlation in evolution that comprise ovarian cancer are close.PTEN expresses and reduces gradually in ovarian cancer generating process, and prompting PTEN genetic expression plays a significant role as early molecule event in ovarian cancer occurs.It is negative correlation that PTEN expresses with the clinicopathologic stage of ovarian cancer, and prompting PTEN expresses decline may be close with the correlation in evolution of ovarian cancer.In addition, in ovarian cancer, PTEN expresses when declining and can shorten without recurrence interval, and prolongation is in progress by PTEN expression rising.(T. U.-J. G.Roth，Time to progression is dependent on theexpression of the tumour suppressor PTEN in ovarian cancer patients.European Journal of ClinicalInvestigation Volume 33，Issue 3，pages 256-260，March 2003；GOMES，C.P.；ANDRADE，L.A.L.A.PTEN and p53 expression in primary ovarian carcinomas：immunohistochemical study anddiscussion of pathogenetic mechanisms.International Journal of Gynecological Cancer：February2006-Volume 16-Issue-p 254-258)

The DNA cloning situation of nonrandomness is there is in karyomit(e) 20q12-q13.2 district in human cancer, many reports are had in ovarian cancer, its high-frequency gene amplification may be relevant to pathogeny, along with tumour progression, its abnormal frequency significantly increases, and in clinical, can be applicable to tumor classification, as the prognostic marker that tumour progression occurs, relevant to poor prognosis.(L.Dimova，A.Yosifova，B.Zaharieva，et，al.Association of 20q13.2 copy number changes with the advancedstage of ovarian cancer-tissue microarray analysis.European Journal of Obstetrics&Gynecologyand Reproductive Biology.Volume 118，Issue 1，10 January 2005，Pages 81-85)

3. ZNF217 (Zinc finger protein 217): being positioned human chromosomal 20q13 district, is a kind of oncogene.ZNF217 gene is the promotion gene of the propagation of ovarian cancer, invasion and attack, motion.There are some researches show, ZNF217 can weaken the apoptotic signal that doxorubicin causes, and hint ZNF217 expresses may be relevant with chemotherapy resistance.ZNF217 by being combined with specific DNA sequence dna, can being formed resistance thing c altogether and holding associated proteins (CtBP) to stop transcribing of several genes.The incorrect expression of ZNF217 causes the negative regulator with cancer cell multiplication, growth and/or Genes of Invasion.ZNF217 gene is expected to the target spot becoming ovarian cancer gene treatment.(Peixiang Li，Sarah Maines-Bandiera，Wen-Lin Kuo.Multiple roles of the candidateoncogene ZNF217 in ovarian epithelial neoplastic progression.International Journal ofCancer.Volume 120，Issue 9，pages 1863-1873，1 May 2007；Kate G.R.Quinlan，Alexis Verger，Paul Yaswen.Amplification of zinc finger gene 217(ZNF217)and cancer：When good fingers gobad.Biochimica et Biophysica Acta(BBA)-Reviews on Cancer.Volume 1775，Issue 2，June 2007，Pages 333-340；Alexander Schipf，Doris Mayr，Thomas Kirchner，et，al.Molecular geneticaberrations of ovarian and uterine carcinosarcomas-a CGH and FISH study.Virchows Archiv，Volume 452，Number 3，259-268.)

4. AURKA (Aurora kinase A, or BTAK, STK15): be positioned human chromosomal 20q13 district.Research shows in Epithelial ovarian tumor proliferation process, and the amplification of AURKA and expression are general and important events, may cause important precancerous lesion.(C.M.Chung，C.Man，Y.Jin.Amplification and overexpression ofAurora kinase A(AURKA)in immortalized human ovarian epithelial(HOSE)cells.MolecularCarcinogenesis.165-174，July 2005)

5. AIB1 (Amplified in breast cancer 1 also claims steroid receptor coactivator-3): be positioned human chromosomal 20q13 district.Steroid kinases coactivator gene, relevant to estrogen receptor positive, with survival of patients difference correlation.(Minna M.Tanner，Seija Grenman，Anjila Koul，et，al.Frequent Amplification of ChromosomalRegion 20q12-q13 in Ovarian Cancer.Clin Cancer Res May 20006；1833)

6. WT1 (Wilms ' tunor suppressor gene): be positioned human chromosomal 11p13 district.Product is considered to the endogenous immunity of tool, thinks now to have carinogenicity.A research shows, in the normal tissue, WT1 only expresses in kidney, spleen slime layer, sustentacular cell of testis, gonad granulocyte.And in ovarian epithelial carcinoma WT1 high expression level, relevant to classification neoplasm staging, with lifetime have nothing to do.Because of its tissue specific expression feature and endogenous immunity, WT1 may become the new target drone of antigen specific immune treatment in ovarian epithelial carcinoma.In addition, WT1 expresses and clinical prognosis difference correlation.(Bonnie Hylandera，Elizabeth Repaskya，Protul Shrikanta，Expression of Wilms tumor gene(WT1)in epithelial ovariancancer，Gynecologic Oncology，Volume 101，Issue 1，April 2006，Pages 12-17；Jakob Dupont，Ekaterina S.Doubrovina，Chia Ma，Development and analysis of Wilms’tumor protein(WT1)specific T cells for adoptive immunotherapy of epithelial ovarian cancer.Proc Amer Assoc CancerRes，Volume 46，2005)

7. PIK3CA (Phosphatidylinositol 3-kinase, catalytic, alpha polypeptide): be positioned human chromosomal 3q26 district.PIK3CA gene copy in about 40% ovary and other cancer increases, its encoding phosphatidylinositol 3-kinases (PI3-kinase) p110 α catalyzer subunit.Because the effect of PI3 kinases in tumor proliferation, glucose metabolism, cytoadherence, apoptosis etc., make PIK3CA participate in tumour as a kind of proto-oncogene and occur.(Laleh Shayesteh，Yiling Lu，Wen-Lin Kuo.PIK3CA is implicated as an oncogene in ovarian cancer.Nature Genetics(1999)21，99-102)

8. MDR1 (multi-drug resistance gene): be positioned human chromosomal 7q21 district.The expression product p-gp of multidrug resistance gene MDR is a kind of energy dependence Teat pipette, medicines different with mechanism of action for various structures can be pumped extracellular, make drug accumulation density loss in born of the same parents, thus hinder performance, its expression level and the drug-resistant intensity positive correlation of chemotherapeutics effect.The combined chemotherapy based on platinum class of current clinical application is better to the result for the treatment of of ovarian cancer, but most of Patients with Advanced Ovarian Carcinoma can recur eventually and produce chemotherapeutics resistance, thus causes Endodontic failure.In vitro study finds, in the Ovarian Cancer Cells to cis-platinum, Adriamycin resistant, the overexpression of MDR1 gene has appearred in the cell of 72%.Suppressed the expression of MDR1 gene by multiple method, not only make cell internalizing treat drug level increase and also enhance the susceptibility of tumour cell to medicine simultaneously.The resistance mechanism of ovarian cancer is very complicated, is the coefficient result of polygene, and detects this target and will contribute to understanding ovarian cancer drug-resistant mechanism further.(Tatyana A.Holzmayer，Susan Hilsenbeck.Clinical Correlates of MDR1P-glycoprotein)Gene Expression in Ovarian and Small-Cell Lung Carcinomas.JNCI J NatlCancer Inst(1992)84(19)：1486-1491.Maria Kavallaris，Jennifer A.Leary，Julie A.et，al.MDR1and multidrug resistance-associated protein(MRP)gene expression in epithelial ovariantumors.Cancer letters，volume102，issues1-2，19 April 1996：7-16.)

Utilize fluorescent probe to detect above-mentioned eight kinds of marks, more comprehensively can be familiar with the mechanism of ovarian cancer, analyze the state of an illness of patient more accurately, in clinical application, understand developing of ovarian cancer.By molecule parting, the prognosis of assessment ovarian cancer patients, is used to guide the formulation of individualized treatment scheme, selects suitable medicine, thus reduces mortality ratio, Economy type medicine cost, reaches the object optimizing treatment effect.

The invention provides the preparation method of the probe of these eight kinds molecular markers relevant to ovarian cancer, and utilize these probe independent developments ovarian cancer molecular marker fluorescence in situ hybridization detection test kit on this basis, fill up the blank of this detection field domestic, there is very great meaning.

Summary of the invention

An object of the present invention is to provide a kind of detection agent of ovarian cancer individualized treatment, detection agent of the present invention comprises BRCA1 and/or PTEN and/or WT1 and/or PIK3CA and/or ZNF217 and/or AURKA and/or AIB1 and/or MDR1 gene probe.

According to the present invention's preferred embodiment, retrieve all clones containing target gene respectively by NCBI Mapview database, respectively these clones are screened, select optimum clone.

According to the present invention's preferred embodiment, this detection agent comprises BRCA1 gene probe: GSP BRCA1

GSP BRCA1 comprises the first probe and the second probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 398Kb beyond BRCA1 gene 5 ' end; Second probe comprises the nucleotide sequence of 102Kb beyond 23Kb and 3 ' end beyond BRCA1 gene and 5 ' end; Described 2 probes are all positioned at 17q21 district.

In one embodiment of the invention, the nucleotide sequence length of 2 probes described in GSP BRCA1 is always 398Kb.Probe is bought in Invitrogen RP11 BAC clone bank.Probe is RP11-948G15 (its Insert Fragment start-stop position: chr17:40981496-41172473, end sequence registration number: AQ571046 and AQ565418) and the Insert Fragment that comprises of RP11-831F13 (its Insert Fragment start-stop position: chr17:41172482-41379594, end sequence registration number: AQ818185 and AQ828457).

According to the present invention's preferred embodiment, this detection agent comprises PTEN gene probe: GSP PTEN

GSP PTEN comprises the first probe, the second probe and the 3rd probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 487Kb beyond PTEN gene 5 ' end; Second probe comprise 61Kb beyond part PTEN gene 98Kb and 5 ' end and nucleotide sequence; 3rd probe comprises the nucleotide sequence of 170Kb beyond part PTEN gene 13Kb and 3 ' end; Described 3 probes are all positioned at 10q23 district.

In one embodiment of the invention, the nucleotide sequence length of 3 probes described in GSP PTEN is always 487Kb.Probe is bought in Invitrogen RP11 BAC and CTD BAC clone bank.Probe is RP11-113H4 (its Insert Fragment start-stop position: chr10:89411341-89571593, end sequence registration number: AQ343267 and AQ343270), CTD-2557P6 (its Insert Fragment start-stop position: chr10:89562163-89721747, end sequence registration number: AQ426941 and AQ426940) and the Insert Fragment that comprises of CTD-2557P6 (its Insert Fragment start-stop position: chr17:89715222-89898644, end sequence registration number: AQ514109 and AQ462258).

According to the present invention's preferred embodiment, this detection agent comprises WT1 gene probe: GSP WT1

GSP WT1 comprises the first probe, the second probe and the 3rd probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 182Kb beyond WT1 gene 5 ' end; Second probe comprise 98Kb beyond WT1 gene and 5 ' end and and 3 ' end beyond the nucleotide sequence of 13Kb; The nucleotide sequence of 188Kb beyond 3rd probe WT1 gene 3 ' end; Described 3 probes are all positioned at 11p13 district.

In one embodiment of the invention, the nucleotide sequence length of 3 probes described in GSP WT1 is always 521Kb.Probe is bought in Invitrogen RP11 BAC and CTD BAC clone bank.Probe is RP11-1037K14 (its Insert Fragment start-stop position: chr11:32136627-32319471, end sequence registration number: AQ693687 and AQ776592), CTD-2643K15 (its Insert Fragment start-stop position: chr11:32310961-32470989, end sequence registration number: AQ588043 and AQ588042) and the Insert Fragment that comprises of RP11-195J14 (its Insert Fragment start-stop position: chr11:32469702-32657875, end sequence registration number: AQ412679 and AZ517572).

According to the present invention's preferred embodiment, this detection agent comprises PIK3CA gene probe: GSP PIK3CA

GSP PIK3CA comprises the first probe, the second probe and the 3rd probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 202Kb beyond PIK3CA gene 5 ' end; Second probe comprise 43Kb beyond PIK3CA gene and 5 ' end and and 3 ' end beyond the nucleotide sequence of 54Kb; 3rd probe is the nucleotide sequence of 104Kb beyond PIK3CA gene 3 ' end; Described 3 probes are all positioned at 3q26 district.

In one embodiment of the invention, the nucleotide sequence length of 3 probes described in GSP PIK3CA is always 477Kb.Probe is bought in Invitrogen RP11 BAC and CTD BAC clone bank.Probe is RP11-737O18 (its Insert Fragment start-stop position: chr3:178633717-178836408, end sequence registration number: AQ450715 and AQ456038), RP11-466H15 (its Insert Fragment start-stop position: chr3:178822841-179006790, end sequence registration number: AQ636573 and AQ636574) and the Insert Fragment that comprises of CTD-2333C22 (its Insert Fragment start-stop position: chr3:179007577-179111560, end sequence registration number: AQ038360 and AQ038356).

According to the present invention's preferred embodiment, this detection agent comprises AIB1 gene probe: GSP AIB1

GSP AIB1 comprises the first probe and the second probe, wherein

First probe nucleic acid sequence comprises the nucleotide sequence of 36Kb beyond AIB1 gene 126Kb and 5 ' end; Second probe comprises the nucleotide sequence of 145Kb beyond AIB1 gene 47Kb and 3 ' end; Described 2 probes are all positioned at 20q13 district.

In one embodiment of the invention, the nucleotide sequence length of 2 probes described in GSP AIB1 is always 335Kb.Probe is bought in Invitrogen RP11 BAC clone bank.Probe is RP11-1151C1 (its Insert Fragment start-stop position: chr20:46094848-46256586, end sequence registration number: AQ749554 and AQ749180) and the Insert Fragment that comprises of RP11-122N8 (its Insert Fragment start-stop position: chr20:46238389-46430555, end sequence registration number: AQ344397 and AZ520185).

According to the present invention's preferred embodiment, this detection agent comprises AURKA gene probe: GSP AURKA

GSP AURKA comprises the first probe, the second probe and the 3rd probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 190Kb beyond AURKA gene 5 ' end; Second probe comprise 56Kb beyond AURKA gene and 5 ' end and and 3 ' end beyond the nucleotide sequence of 99Kb; The nucleotide sequence of 176Kb beyond 3rd probe AURKA gene 3 ' end; Described 3 probes are all positioned at 20q13 district.

In one embodiment of the invention, the nucleotide sequence length of 3 probes described in GSPAURKA is always 497Kb.Probe is bought in Invitrogen RP11 BAC and CTD BAC clone bank.Probe is RP11-688E22 (its Insert Fragment start-stop position: chr20:54730219-54921077, end sequence registration number: AQ808759 and AQ813272), RP11-1067D15 (its Insert Fragment start-stop position: chr20:54888353-55066415, end sequence registration number: AQ730353

And AQ730207) and the Insert Fragment that comprises of CTD-2592H20 (its Insert Fragment start-stop position: chr20:55051190-55227192, end sequence registration number: AQ476668 and AQ476665).

According to the present invention's preferred embodiment, this detection agent comprises ZNF217 gene probe: GSP ZNF217

GSP ZNF217 comprises the first probe and the second probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 152Kb beyond ZNF217 gene 5 ' end; Second probe comprises the nucleotide sequence of 125Kb beyond 54Kb and 3 ' end beyond ZNF217 gene and 5 ' end; Described 2 probes are all positioned at 20q13 district.

In one embodiment of the invention, the nucleotide sequence length of 2 probes described in GSP ZNF217 is always 337Kb.Probe is bought in Invitrogen RP11 BAC clone bank.Probe is RP11-91L1 (its Insert Fragment start-stop position: chr20:51987810-52139422, end sequence registration number: AZ519337 and AQ283579) and the Insert Fragment that comprises of RP11-1057P5 (its Insert Fragment start-stop position: chr20:52129960-52324790, end sequence registration number: AQ673688 and AQ680772).

According to the present invention's preferred embodiment, this detection agent comprises MDR1 gene probe: GSP MDR1

GSP MDR1 comprises the first probe, the second probe and the 3rd probe, wherein

First probe nucleic acid sequence is the nucleotide sequence of the 155Kb beyond MDR1 gene 5 ' end; Second probe be beyond MDR1 gene and 5 ' end beyond 51Kb and 3 ' end 3Kb nucleotide sequence; The nucleotide sequence of 171Kb beyond 3rd probe MDR1 gene 3 ' end; Described 3 probes are all positioned at 7q21 district.

In one embodiment of the invention, the nucleotide sequence length of 3 probes described in GSP MDR1 is always 473Kb.Probe is bought in Invitrogen RP11 BAC clone bank.Probe is RP11-47N1 (its Insert Fragment start-stop position: chr7:87037167-87192745, end sequence registration number: AQ200820 and AQ200819), RP11-1067D15 (its Insert Fragment start-stop position: chr20:87177953-87345695, end sequence registration number: AQ315289 and AQ315285) and the Insert Fragment that comprises of CTD-2592H20 (its Insert Fragment start-stop position: chr20:87338969-8750978, end sequence registration number: B71252 and AZ517192).

The clone using screening to obtain prepares said gene probe, and fluorescence in situ hybridization test result shows, and hybridization signal becomes clear, and can be used for pattern detection.

According to the present invention's preferred embodiment, in gene probe preparation process, cloned plasmids DNA extraction can use commercially available plasmid extraction kit, includes but not limited to, as the Plasmid Maxi Kit of Qiagen company.

According to the present invention's preferred embodiment, in gene probe preparation process, cloned plasmids DNA's is quantitatively the absorbancy measured respectively after plasmid DNA being diluted under 260nm and 280nm, calculates production concentration.

According to the present invention's preferred embodiment, the mark of gene probe can adopt method of the prior art by corresponding fluorescein-labelled on double-strandednucleic acid, described method includes but not limited to: random priming, nick translation etc., mark gene probe can use commercially available nick translation labelling kit and/or random primer labelling kit, the Nick Translation Kit of preferred abbott and/or Roche company.

According to the present invention's preferred embodiment, the fluorescein of Probe labelling can select fluorescence dye known in the art, as: Alexa 488, Alexa 555, pacific fITC, DEAC, Rhodamine etc., the different probe in same gene probe groups, as BRCA1 gene probe: the first probe and the second probe in GSP BRCA1, use same fluorescence dye to mark in principle.

According to the present invention's preferred embodiment, when carrying out multi objective joint-detection, in multicolor fluorescence system, preferred isothiocyanic acid (green) and/or rhodamine (redness) and/or DEAC (cyan) and/or spectrum Gold (gold) carry out composite marking.

According to the present invention's preferred embodiment, when carrying out single index and detecting, in fluorescence system, preferred isothiocyanic acid (green) or rhodamine (redness) mark.

According to the present invention's preferred embodiment, for gene probe, when marking with rhodamine, under normal circumstances, 2 danger signals (2R) should be shown under fluorescent microscope; When there is homozygous mutant gene, under fluorescent microscope, do not show danger signal (0R); When there is gene amplification, under fluorescent microscope, show multiple danger signal (> 2R).

Another object of the present invention utilizes BRCA1 and/or PTEN and/or WT1 and/or PIK3CA and/or ZNF217 and/or AURKA and/or AIB1 and/or MDR1 gene probe to prepare a kind of ovarian cancer molecular marker fluorescence in situ hybridization detection test kit.

In order to realize the present invention, we have employed following technical scheme:

(1) sample process and film-making: all types of sample is processed according to in-situ hybridization method.

(2) hybridize: preparing hybrid liquid, mainly comprises fluorescence labeling probe mixture and hybridization buffer, carries out the hybridization of 8 ~ 16 hours under suitable temp, then wash away with suitable washing lotion that do not combine with probe that is non-specific binding; DAPI counterstain is redyed.

(3) observe fluorescent signal by the corresponding filter block of fluorescent microscope, observation counting is carried out to unlike signal.

The test kit invented based on above technical scheme comprises: 1) hybridization buffer, fluorescence labeling probe mixture, unlabelled competitive DNA, DAPI counterstain, and 2) separate and concentrate the packing box packing these reagent bottles or pipe.

According to a preferred embodiment of the invention, the component of hybridization buffer comprises deionized formamide, SSC, T 500, and wherein deionized formamide concentration is 50% ~ 70%, and T 500 concentration is 0.1g/ml.

According to a preferred embodiment of the invention, fluorescence labeling probe mixture comprises BRCA1 and/or PTEN and/or WT1 and/or PIK3CA and/or ZNF217 and/or AURKA and/or AIB1 and/or MDR1 gene probe.

According to a preferred embodiment of the invention, 8 kinds of gene probe groups can be combined according to testing goal, after marking different fluorescence dye respectively, detect for ovarian cancer molecular marker; In every person-portion fluorescence labeling probe mixture, the usage quantity of each gene probe is 0.2 μ l.

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-1 DNA; Add 7 μ l hybridization buffers in every person-portion hybridization solution, 0.2 ~ 0.8 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, the fluorescence in-situ hybridization method according to routine carries out signal-count to the mid-term of people and Interphase cells.

The present invention compared with prior art, has following advantages:

(1) achieve and carry out nearly detecting while 4 kinds of molecular markers to same sample, be conducive to improving the early stage of ovarian cancer

Recall rate, carries out ovarian cancer molecule parting more accurately;

(2) signal-count and result is reproducible is accurately and rapidly carried out;

(3) without the need to carrying out cell cultures and the high-quality metaphase chromosome body piece of preparation, can be used for mid-term or Interphase cells signal-count, operating relatively simple;

(4) by being prepared into test kit, can be implemented in the application in the field such as oncobiology, cytogenetics, help each molecular marker of comprehensive evaluation, that understands that itself and ovarian cancer occur to develop etc. contacts, and the medication of adjuvant clinical targeted therapy and treatment plan are selected.

Accompanying drawing explanation

Fig. 1 shows GSP BRCA1 probe groups location pattern figure (red wire frame probe position).

Fig. 2 shows GSP PTEN probe groups location pattern figure (red wire frame probe position).

Fig. 3 shows GSPWT1 probe groups location pattern figure (red wire frame probe position).

Fig. 4 shows GSP ZNF217 probe groups location pattern figure (red wire frame probe position).

Fig. 5 shows GSP PIK3CA probe groups location pattern figure (red wire frame probe position).

Fig. 6 shows GSP AURKA probe groups location pattern figure (red wire frame probe position).

Fig. 7 shows GSP AIB1 probe groups location pattern figure (red wire frame probe position).

Fig. 8 shows GSP MDR1 probe groups location pattern figure (red wire frame probe position).

Fig. 9 shows the detected result of mankind's proper splitting lymphocyte in mid-term gene probe.

Figure 10 shows the detected result of mankind's proper splitting lymphocyte in mid-term combination gene probe (dichromatism).

Figure 11 shows the detected result of mankind's proper splitting lymphocyte in mid-term combination gene probe (four looks).

Figure 12 shows the detected result that gene probe (four looks) in paraffin-embedded tissue section fixed by ovarian cancer tissue's formalin.

Embodiment

The following example is intended to illustrate instead of limit the present invention.

Embodiment 1: the preparation method of ovarian cancer molecular marker detection agent BRCA1 gene probe

(1) colony screening: select and comprise goal gene BRCA1 and two terminal sequences are the clone of RP11-948G15 and RP11-831F13.

(2) clone culture & identification: buy corresponding clone Invitrogen RPCI11.C, get 50ul and add in the TB nutrient solution (chlorampenicol resistant) of 500ml, shake bacterium in 37 DEG C of shaking tables and cultivate 24 ~ 48 hours; Bacterium liquid uses STS primer pair to carry out clone identification.RP11-948G15:STS primer pair: upstream primer 5 '-CTTGCACCTATAATCCCAG-3 ' and downstream primer 5 '-AGTCTTCTTGTCTTGTGGC-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 128bps has bright band.RP11-831F13STS primer pair: upstream primer 5 '-AAACATGTTCCTCCTAAGGTGCTTT-3 ' and downstream primer 5 '-ATGAAACCAGAAGTAAGTCCACCAGT-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 119bps has bright band.

(3) GSP BRCA1 gene probe preparation: the bacterium liquid that qualification is positive, use the Plasmid Maxi Kit of Qiagen company, the working method required to specifications carries out ultralow copy extraction of plasmid DNA, quantitative to plasmid DNA by the absorbancy measuring 260nm and 280nm place; According to formula: Double stranded DNA concentration (ng/ul)=0D260 × 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water to dilute for 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 DEG C of sealings are preserved.

Carry out fluorescent mark by nick translation method to plasmid DNA, the fluorescein of probe mark is FITC-12-dUTP.Adopt the Nick Translation Kit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.

Join rear concussion mixing, mark 12 hours at 16 DEG C, then 80 DEG C hatch 10 minutes inactivators.Getting 5ul uses 2% sepharose to do electrophoresis, requires the band that there is disperse between 100-500bp.

Alcohol settling carried out to marked product and concentrate, in 1.5ml centrifuge tube, adding sodium-acetate and dehydrated alcohol by following scheme successively, lucifuge, on ice preparation:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing to be placed in-70 DEG C of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 DEG C of 13000rpm, carefully remove supernatant, do not stir precipitation, add 70% ethanol of 1ml, 4 DEG C 13000 revs/min centrifugal 15 minutes, carefully removes supernatant, do not stir precipitation, and lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP BRCA1 gene probe, lucifuge ,-20 DEG C of storages.

(4) GSP BRCA1 gene probe checking: use mankind's proper splitting lymphocyte in mid-term to drip sheet and carry out probe checking.Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.

Embodiment 2: the preparation method of ovarian cancer molecular marker detection agent PTEN gene probe

(1) colony screening: select and comprise goal gene PTEN and two terminal sequences are the clone of RP11-113H4, CTD-2557P6 and RP11-765C10.

(2) clone culture & identification: RP11-113H4:STS primer pair: upstream primer 5 '-AGAAAGACAGACAAAAAGATGAGG-3 ' and downstream primer 5 '-GAGTTGCCAGTGTGCTTTAC-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 188bps has bright band.CTD-2557P6 STS primer pair: upstream primer 5 '-GTTTTTAGTGAGGGCTGGTGAAA-3 ' and downstream primer 5 '-ATTTCCCTTTGGAAAGTGCTGTT-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 341bps has bright band.RP11-765C10STS primer pair: upstream primer 5 '-TGTTAAGCTTGTCTATGCTAAACAA-3 ' and downstream primer 5 '-AATTCAGTGGCTTAATCATGAATG-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 341bps has bright band.Other step is see embodiment 1.

(3) GSP PTEN gene probe preparation: the bacterium liquid that qualification is positive, use the Plasmid Maxi Kit of Qiagen company, the working method required to specifications carries out ultralow copy extraction of plasmid DNA, quantitative to plasmid DNA by the absorbancy measuring 260nm and 280nm place; According to formula: Double stranded DNA concentration (ng/ul)=OD260 × 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water to dilute for 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 DEG C of sealings are preserved.

Carry out fluorescent mark by nick translation method to plasmid DNA, the fluorescein of probe mark is Spectrum orange (or gold) dUTP.Adopt the Nick Translation Kit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.

Join rear concussion mixing, mark 12 hours at 16 DEG C, then 80 DEG C hatch 10 minutes inactivators.Getting 5ul uses 2% sepharose to do electrophoresis, requires the band that there is disperse between 100-500bp.

Alcohol settling carried out to marked product and concentrate, in 1.5ml centrifuge tube, adding sodium-acetate and dehydrated alcohol by following scheme successively, lucifuge, on ice preparation:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing to be placed in-70 DEG C of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 DEG C of 13000rpm, carefully remove supernatant, do not stir precipitation, add 70% ethanol of 1ml, 4 DEG C 13000 revs/min centrifugal 15 minutes, carefully removes supernatant, do not stir precipitation, and lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP BRCA1 gene probe, lucifuge ,-20 DEG C of storages.

(4) GSP PTEN gene probe checking: use mankind's proper splitting lymphocyte in mid-term to drip sheet and carry out probe checking.Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.

Embodiment 3: the preparation method of ovarian cancer molecular marker detection agent WT1 gene probe

(1) colony screening: select and comprise goal gene WT1 and two terminal sequences are the clone of RP11-1037K14, CTD-2643K15 and RP11-195J14.

(2) clone culture & identification: RP11-1037K14:STS primer pair: upstream primer 5 '-AGCTCAAGTGGACAGATGTACAGG-3 ' and downstream primer 5 '-TGCAATTAGCACGACCATGATAC-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 323bps has bright band.CTD-2643K15STS primer pair: upstream primer 5 '-GTTGTCCTTTTCAGCATTGC-3 ' and downstream primer 5 '-GCAGGGTTTCATCCTCGG-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 405bps has bright band.RP11-195J14STS primer pair: upstream primer 5 '-CCTTTGCTTATGCTGCTTCC-3 ' and downstream primer 5 '-CATGCTTCATGCTTCTCTATGG-3 ', pcr amplification condition is: 95 DEG C 5 minutes; (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 45 seconds) × 35 circulations; 72 DEG C 7 minutes.Amplified production carries out electrophoresis checking, and about result 130bps has bright band.Other step is see embodiment 1.

(3) GSP WT1 gene probe preparation: the bacterium liquid that qualification is positive, use the Plasmid Maxi Kit of Qiagen company, the working method required to specifications carries out ultralow copy extraction of plasmid DNA, quantitative to plasmid DNA by the absorbancy measuring 260nm and 280nm place; According to formula: Double stranded DNA concentration (ng/ul)=OD260 × 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water to dilute for 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 DEG C of sealings are preserved.

Carry out fluorescent mark by nick translation method to plasmid DNA, the fluorescein of probe mark is DEAC-dUTP.Adopt the Nick Translation Kit of abbott, by following scheme, under strict lucifuge condition, prepare PCR reaction system on ice.

Join rear concussion mixing, mark 12 hours at 16 DEG C, then 80 DEG C hatch 10 minutes inactivators.Getting 5ul uses 2% sepharose to do electrophoresis, requires the band that there is disperse between 100-500bp.

Alcohol settling carried out to marked product and concentrate, in 1.5ml centrifuge tube, adding sodium-acetate and dehydrated alcohol by following scheme successively, lucifuge, on ice preparation:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing to be placed in-70 DEG C of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 DEG C of 13000rpm, carefully remove supernatant, do not stir precipitation, add 70% ethanol of 1ml, 4 DEG C 13000 revs/min centrifugal 15 minutes, carefully removes supernatant, do not stir precipitation, and lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP WT1 gene probe, lucifuge ,-20 DEG C of storages.

(4) GSP WT1 gene probe checking: use mankind's proper splitting lymphocyte in mid-term to drip sheet and carry out probe checking.Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus in form.

Embodiment 4: the preparation method of ovarian cancer molecular marker detection agent GSP PIK3CA, GSP ZNF217, GSP AURKA, GSP AIB1, GSP MDR1 gene probe

(1) clone and the list of STS primer pair

Table 1 GSP PIK3CA probe screening and cloning and STS primers designed pair

Table 2 GSP ZNF217 probe screening and cloning and STS primers designed pair

Table 3 GSP AURKA probe screening and cloning and STS primers designed pair

Table 4 GSP AIB1 probe screening and cloning and STS primers designed pair

Table 5 GSP MDR1 probe screening and cloning and STS primers designed pair

Note: primer sequence is 5` to 3`.

(2) probe preparation and verification method are with embodiment 1 ~ 3.

Embodiment 5: human ovarian cancer molecular marker fluorescence in situ hybridization detection test kit preparation (dichromatism)

For 10 person-portions/box.

(1) hybridization solution preparation

The probe marked is put well successively, first dissolves probe.Use 1 μ l sterilizing purified water to dissolve in the gene probe dry powder be prepared by embodiment 1 ~ 4 method, fully mix.By table 6 scheme preparation fluorescence labeling probe mixture:

Table 6

According to the form below scheme preparing hybrid liquid:

Table 7

(2) DAPI counterstain preparation

The anti-liquid that fades: the necessary lucifuge of substantial length of operation, the Ursol D of 10mg is dissolved in the PBS of 1ml, regulates pH to be 9.0, adds 9ml glycerine, repeatedly shake mixing ,-20 DEG C of storages.Whole solution should be colourless or slightly faint yellow, if present yellow or orange, needs abandoned well again to prepare.

By deionized water preparation 1mg/ml DAPI storage liquid.

The DAPI solution (0.1mg/ml) getting 2.5 μ l is dissolved in that 1ml is anti-to fade in liquid, repeatedly shakes mixing ,-20 DEG C of airtight preservations of lucifuge under lucifuge condition.

(3) finished product assembling

Ingredient names	Specification	Quantity
			Hybridization solution 1,2,3,4	Each 100 μ l/ manage	4 pipes
DAPI counterstain	400 μ l/ manage	1 pipe
			Specification sheets		1 part

Detected result as shown in Figure 10.

Embodiment 6: human ovarian cancer molecular marker fluorescence in situ hybridization detection test kit preparation (four looks)

For 10 person-portions/box.

(4) hybridization solution preparation

The probe marked is put well successively, first dissolves probe.Use 1 μ l sterilizing purified water to dissolve in the gene probe dry powder be prepared by embodiment 1 ~ 4 method respectively, fully mix.According to the form below scheme preparation fluorescence labeling probe mixture:

Table 8

According to the form below scheme preparing hybrid liquid:

Table 9

(5) DAPI counterstain preparation

With embodiment 5.

(6) finished product assembling

Ingredient names	Specification	Quantity
			Hybridization solution 1,2	Each 100 μ l/ manage	2 pipes
DAPI counterstain	200 μ l/ manage	1 pipe
			Specification sheets		1 part

Detected result as shown in figure 11.

Embodiment 7: the using method of ovarian cancer molecular marker fluorescence in situ hybridization detection test kit (four looks)

The ovary tissue sample that paraffin buries is fixed for formalin.

(1) slide pre-treatment

Slide is put into the roasting sheet of 65 ± 5 DEG C of thermostat containers and is spent the night; Take out slide, put into room temperature dimethylbenzene 30 minutes, put it into the dimethylbenzene of room temperature 1: 1 (V: V) again: in dehydrated alcohol 10 minutes, put into room temperature dehydrated alcohol 10 minutes, then put into room temperature 100% ethanol, 90% ethanol, each 3 minutes of 70% ethanol successively; Leave standstill 3 minutes in deionized water at room temperature, draw excessive moisture; (section was placed horizontally in container in 25 minutes to boil sheet in the deionized water of 100 ± 5 DEG C, sample faces up), after drying, put into the Proteinase K reaction solution digestion about 8 ~ 15 minutes of 37 ± 1 DEG C of preheatings, rinsing 5 minutes in room temperature 2 × SSC, put into room temperature 70% successively, 90%, 100% each 3 minutes of graded ethanol dehydration.Dry slide; Continue crossover process.

(2) sample and the same time variation of probe

Hybridization solution is taken out, concussion mixing, brief centrifugation from test kit; Add the hybridization solution of 10 μ l to hybridising region, rapid covered, light pressure makes hybridization solution be uniformly distributed, and avoids producing bubble; Rubber cement, along cover glass edge mounting, covers the edge that cover glass contacts with slide glass completely; Sex change 5 minutes in the thermal station of 85 ± 1 DEG C; Slide is put into the hybridizing box of preheating, lucifuge, 37 ± 1 DEG C of overnight incubation (about 16 hours); Continue post-hybridization washing step.

(3) post-hybridization washing and redying

Take out the hybridizing box of overnight incubation, careful removal rubber cement and cover glass; Slide is put into 37 ± 1 DEG C of 2 × SSC 10 minutes; Put it into 37 ± 1 DEG C of 0.1%NP-40/2 × SSC again and wash 2 minutes; Room temperature 70% ethanol 3 minutes; Dark place is dried.

Redye: drip 10 μ l DAPI to slide glass target area, covered, gently presses, avoid producing bubble, in the dark deposit, to be seen.

(4) interpretation of result

Under Olympus BX50 fluorescent microscope, observe by filter group the hybridization fluorescent signal that DAPI redyes respectively, use CCD photographic recording.Find under 40 × object lens, count under 100 × object lens; Adjust suitable focal length, have clear and definite concept to signal and background; Signaling point is because being positioned at cell; When extracellular exists fluorescent signal point, note distinguishing with Intracellular signals point, preferably can avoid this region and count; Adjusting focal length, finds signaling point in the different levels of core; The number of each probe in each cell observed in record.

(5) result judges

Operate by treatment process requirement, record the signal number of each gene probe.Fluorescence in situ hybridization result shows, and on Metaphase Chromosome, all visible two signaling points of each gene probe, have no other fluorescent signal; Rarely seen two signaling points in interphase nuclei, have no other fluorescent signal (see accompanying drawing 12).

Embodiment 8: the Clinical practice evaluation of ovarian cancer molecular marker fluorescence in situ hybridization detection test kit

The routine ovarian cancer sample of clinical collection 20, uses ovarian cancer fluorescence in situ hybridization detection test kit described in the present embodiment 7 to detect respectively, carries out operation and result judgement according to embodiment 7.Detected result is as described in Table 10.

Table 10 clinical sample case information

Table 11 ovarian cancer molecular marker detected result

Note: normal N; Amplification A; Disappearance D.

Detected result is explained:

1. BRCA1 disappearance prompting ovarian tumor occurs.

2. PTEN disappearance points out generation and the evolution of ovarian tumor, may be early molecule event during ovarian cancer occurs.

3. WT1 amplification is relevant to classification neoplasm staging, may become the new target drone of antigen specific immune treatment in ovarian epithelial carcinoma, with clinical prognosis difference correlation.

4. ZNF217 amplification prompting human epithelial ovarian carcinoma cells proliferation, growth and/or invasion and attack occur, can as the target spot of targeted therapy.

5. PIK3CA participates in tumour generation, amplification prompting ovarian cancer.

6. AURKA amplification is event general and important in ovarian cancer, may cause important precancerous lesion.

7. AIB1 amplification indication patient poor prognosis.

8. MDR1 amplification prompting platinum class resistance, measurable curative effect and toxic side effect.

Know from detected result, after molecular marker detection is carried out to these samples, molecule parting can be carried out according to detected result to sample, according to the meaning of Testing index, the stage and step of ovarian cancer can be assisted, meanwhile, for clinical treatment formulation, medication selection and Outcome measure.

Therefore, by carrying out the joint-detection of this eight point date to doubtful ovarian cancer tissue sample, help each molecular marker of comprehensive evaluation, understand contacting of itself and tumour generation etc.Simultaneously for clear and definite gene Clustering, instruct clinical application/treatment and judging prognosis effect all extremely useful.

Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.

Claims (5)

1. an ovarian cancer molecular marker detection kit, comprise: 1) hybridization solution, DAPI counterstain, with 2) separate and concentrate the packing box packing these reagent bottles or pipe, wherein hybridization solution is formulated by hybridization buffer, fluorescently-labeled gene probe and closed DNA; And described gene probe is made up of following probe:

(1)GSP BRCA1

Be made up of the first probe and the second probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 398Kb beyond BRCA1 gene 5 ' end; Second probe is made up of the nucleotide sequence of 102Kb beyond 23Kb and 3 ' end beyond BRCA1 gene and 5 ' end; Described 2 probes are all positioned at 17q21 district;

(2)GSP PTEN

Be made up of the first probe, the second probe and the 3rd probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 487Kb beyond PTEN gene 5 ' end; Second probe holds the nucleotide sequence of 61Kb beyond the part PTEN gene of 98Kb and 5 ' end to form by 5 '; 3rd probe is made up of the nucleotide sequence of 170Kb beyond part PTEN gene 13Kb and the 3 ' end of 3 ' end 13Kb; Described 3 probes are all positioned at 10q23 district;

(3)GSP WT1

Be made up of the first probe, the second probe and the 3rd probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 182Kb beyond WT1 gene 5 ' end; Second probe is made up of the nucleotide sequence of 13Kb beyond 98Kb and 3 ' end beyond WT1 gene and 5 ' end; 3rd probe is the nucleotide sequence of 188Kb beyond WT1 gene 3 ' end; Described 3 probes are all positioned at 11p13 district;

(4)GSP PIK3CA

Be made up of the first probe, the second probe and the 3rd probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 202Kb beyond PIK3CA gene 5 ' end; Second probe is made up of the nucleotide sequence of 54Kb beyond 43Kb and 3 ' end beyond PIK3CA gene and 5 ' end; 3rd probe is the nucleotide sequence of 104Kb beyond PIK3CA gene 3 ' end; Described 3 probes are all positioned at 3q26 district;

(5)GSP AIB1

Be made up of the first probe and the second probe, wherein, the first probe nucleic acid sequence is made up of the nucleotide sequence of 36Kb beyond 5 ' end AIB1 gene 126Kb and 5 ' end; Second probe is made up of the nucleotide sequence of 145Kb beyond 3 ' end AIB1 gene 47Kb and 3 ' end; Described 2 probes are all positioned at 20q13 district;

(6)GSP AURKA

Be made up of the first probe, the second probe and the 3rd probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 190Kb beyond AURKA gene 5 ' end; Second probe is made up of the nucleotide sequence of 99Kb beyond 56Kb and 3 ' end beyond AURKA gene and 5 ' end; 3rd probe is the nucleotide sequence of 176Kb beyond AURKA gene 3 ' end; Described 3 probes are all positioned at 20q13 district;

(7)GSP ZNF217

Be made up of the first probe and the second probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 152Kb beyond ZNF217 gene 5 ' end; Second probe is made up of the nucleotide sequence of 125Kb beyond 54Kb and 3 ' end beyond ZNF217 gene and 5 ' end; Described 2 probes are all positioned at 20q13 district;

(8)GSP MDR1

Be made up of the first probe, the second probe and the 3rd probe, wherein, the first probe nucleic acid sequence is the nucleotide sequence of the 155Kb beyond MDR1 gene 5 ' end; Second probe is made up of the nucleotide sequence of 3Kb beyond 51Kb and 3 ' end beyond MDR1 gene and 5 ' end; 3rd probe is the nucleotide sequence of 171Kb beyond MDR1 gene 3 ' end; Described 3 probes are all positioned at 7q21 district;

Different fluorochrome labels selected by above-mentioned probe.

2. detection kit according to claim 1, it is characterized in that using the amount of gene probe for 0.2ul in every person-portion hybridization solution, hybridization buffer is 7ul, hands over deionized formamide concentration in damping fluid to be 50% ~ 70%.

3. detection kit according to claim 1, is further characterized in that described gene probe is to comprise the clone insert of detection target gene and periphery nucleotide sequence as probe preparation template, adopts random primer or nick-translation method to carry out fluorescent mark.

4. detection kit according to claim 3, is further characterized in that the preparation template of gene probe is for clone combination as follows:

(1)GSP BRCA1：RP11-948G15、RP11-831F13；

(2)GSP PTEN：RP11-113H4、CTD-2557P6、CTD-2557P6；

(3)GSP WT1：RP11-1037K14、CTD-2643K15、RP11-195J14；

(4)GSP PIK3CA：RP11-737018、RP11-466H15、CTD-2333C22；

(5)GSP AIB1：RP11-1151C1、RP11-122N8；

(6)GSP AURKA：RP11-688E22、RP11-1067D15、CTD-2592H20；

(7)GSP ZNF217：RP11-91L1、RP11-1057P5；

(8)GSP MDR1：RP11-47N1、RP11-1067D15、CTD-2592H20。

5. test kit according to claim 1, is further characterized in that detected sample is tissue samples or cell sample.