CN103131757A - 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 the ovarian cancer individualized treatment, comprise the preparation of the related molecule sign detection probes such as genesis, stage and step, prediction curative effect.The ovarian cancer molecular marker detection probes that relates in the present invention can be used for instructing the solution formulation of ovarian cancer individualized treatment, estimates result for the treatment of, monitoring recurrence and transfer, evaluate its prognosis etc.

Background technology

Ovarian cancer is the malignant tumour that betides ovary tissue, and sickness rate is only second to cervical cancer and carcinoma of uterine body, accounts for the 3rd of women's malignant tumour.Because ovarian tumor is positioned at pelvic cavity, at few reveal any symptoms of ill initial stage, morbidity is hidden, progress is rapid, and 70%～80% ovarian cancer patients has been late period when finding, survival rate was only 20%～30% in 5 years, and early ovarian cancer patient's survival rate can reach 90%.At present, it is clinical that generally to carry out to perform the operation be main complex therapy, but due to illness the reason classification is many, complex structure, biological characteristics difference is large, and different to the susceptibility of chemotherapy, radiotherapy etc., causes the difficulty in processing and affects the treatment, its mortality ratio surpasses cervical cancer and carcinoma of uterine body sum, accounts for the first place of gynecological tumor.Therefore, only be improved the early diagnosis level of ovarian cancer, the biological nature of all types of malignants tumor of ovary of grasp, explore effective composite treatment, can strive for favourable treatment opportunity, improve prognosis, improve survival rate, the reduction mortality ratio of ovarian cancer.

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

Targeted therapy can improve medicine to the lethality of tumour cell as a kind of new oncotherapy means, and reduces simultaneously the undesirable action of normal tissue organ, demonstrates clear superiority than the traditional treatment mode, progressively is applied to clinical treatment.Develop rapidly along with basic subjects such as molecular biology, immunology, virusology and genetically engineereds, and people's going deep into tumor development research, targeted therapy as a kind of new treatment pattern with its safety, efficient characteristic, for new field has been started in the treatment of 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 studies show that there are obvious individual difference in different treatment plans and curative effect of medication, utilize the molecular Biological Detection assess patient, formulate treatment plan according to molecule parting, carry out the individuation diagnosis and treatment, for improving treatment effect, reduce mortality ratio, reduce medical treatment cost and all have meaning (Albertson, D.G., 2006.Gene amplification in cancer.Trends Genet.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 Of Medicine 2004, volume71, number 7.).At present, the genetic marker relevant to ovarian cancer, genesis genes involved and the gene studies of medication outcome prediction are more, but mostly rest on the laboratory study stage, lack corresponding molecular diagnosis agents on market, have greatly limited the development of individuation diagnosis and treatment.

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

1. BRCA1 (breast cancer susceptibility gene 1): be positioned human chromosomal 17q21 district.Breast cancer susceptibility gene, cancer suppressor gene, its cancer suppressor protein of encoding participates in many important cell activities, as Control of cellcycle, transcription activating and inhibition, 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, the gene product effect disappears or reduces, and just may cause the generation of ovarian tumor.Studies show that, BRCA1 is high expression level in normal ovarian tissue, express in ovarian cancer and reduce, and along with the cancer pathology histological grading increases and when lymphatic metastasis occurs, positive expression rate descend gradually (Joshua Z Press, Alessandro De Luca, Niki Boyd, et, al.Ovarian carcinomas with genetic and epigenetic 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 cancer susceptibility gene BRCA1.1994,266 (5182): 66-71).

2. PTEN (Phosphatase and tensin homolog): be positioned human chromosomal 10q23 district.PTEN is that first that find up to now has the cancer suppressor gene of phosphatase activity, play a part complicatedly and important in the cell signal transmission of somatomedin, the effect of the performance cancer suppressor genes such as cell mitogen, cell death inducing, participation are regulated the sticking of cell by suppressing, transfer and differentiation.The disappearance of PTEN and sudden change and kinds of tumors comprise that the generation of ovarian cancer and correlation in evolution are close.PTEN expresses in the ovarian cancer generating process and reduces gradually, and prompting PTEN genetic expression plays a significant role as the early molecule event in ovarian cancer occurs.PTEN expresses and the clinicopathologic stage of ovarian cancer is negative correlation, and prompting PTEN expresses decline may be close with the correlation in evolution of ovarian cancer.In addition, in ovarian cancer, PTEN expresses when descending and can shorten without recurrence interval, and PTEN expression rising will extend progress.(T.

U.-J.

G.Roth，Time?to?progression?is?dependent?on?the?expression?of?the?tumour?suppressor?PTEN?in?ovarian?cancer?patients.European?Journal?of?Clinical?Investigation?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?and?discussion?of?pathogenetic?mechanisms.International?Journal?of?Gynecological?Cancer：February?2006-Volume?16-Issue-p?254-258)

There is the DNA cloning situation of nonrandomness in karyomit(e) 20q12-q13.2 district in human cancer, many reports are arranged 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 the tumour somatotype, as the prognosis sign 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?advanced?stage?of?ovarian?cancer-tissue?microarray?analysis.European?Journal?of?Obstetrics&Gynecology?and?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.The ZNF217 gene is propagation, the invasion and attack of ovarian cancer, the promotion gene of motion.There are some researches show, ZNF217 can weaken the apoptotic signal that the Zorubicin treatment causes, and hint ZNF217 expresses may be relevant with the chemotherapy resistance.ZNF217 may by be combined with specific DNA sequence dna, form and hinder altogether thing c end transcribing in conjunction with albumen (CtBP) prevention several genes.The incorrect expression of ZNF217 causes the negative regulator with cancer cell multiplication, growth and/or Genes of Invasion.The ZNF217 gene is expected to become the target spot of ovarian cancer gene treatment.(Peixiang?Li，Sarah?Maines-Bandiera，Wen-Lin?Kuo.Multiple?roles?of?the?candidate?oncogene?ZNF217?in?ovarian?epithelial?neoplastic?progression.International?Journal?of?Cancer.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?go?bad.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?genetic?aberrations?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.Studies show that in the Epithelial ovarian tumor during proliferation process, 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?of?Aurora?kinase?A(AURKA)in?immortalized?human?ovarian?epithelial(HOSE)cells.Molecular?Carcinogenesis.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 the survival of patients difference correlation.(Minna?M.Tanner，Seija?Grenman，Anjila?Koul，et，al.Frequent?Amplification?of?Chromosomal?Region?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.One studies show that, in healthy tissues, WT1 only expresses in kidney, spleen slime layer, sustentacular cell of testis, gonad granulocyte.And in ovarian epithelial carcinoma the WT1 high expression level, relevant to classification neoplasm staging, irrelevant with lifetime.Because of its tissue specific expression characteristics and endogenous immunity, WT1 may become the new target drone of antigen-specific immunotherapy in ovarian epithelial carcinoma.In addition, WT1 expresses and the clinical prognosis difference correlation.(Bonnie?Hylandera，Elizabeth?Repaskya，Protul?Shrikanta，Expression?of?Wilms?tumor?gene(WT1)in?epithelial?ovarian?cancer，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?Cancer?Res，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 coding phosphatidyl-inositol 3-kinase (PI3-kinase) p110 α catalyzer subunit.Because the effect of PI3 kinases in tumor proliferation, glucose metabolism, cytoadherence, apoptosis etc. makes PIK3CA participate in tumour as a kind of proto-oncogene and occurs.(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, can pump the extracellular by the medicine that various structures is different with mechanism of action, make drug accumulation density loss in born of the same parents, thereby hinder performance, its expression level and the drug-resistant intensity positive correlation of chemotherapeutics effect.At present clinical application better to the result for the treatment of of ovarian cancer take the platinum class as the combined chemotherapy on basis, but most of Patients with Advanced Ovarian Carcinoma can recur and produce the chemotherapeutics resistance eventually, thus cause treating unsuccessfully.The in vitro study discovery, the overexpression of MDR1 gene has appearred in 72% cell in to the Ovarian Cancer Cells of cis-platinum, Adriamycin resistant.Suppress the expression of MDR1 gene by several different methods, not only make cell internalizing treat the drug level increase and also strengthened the susceptibility of tumour cell to medicine simultaneously.The resistance mechanism of ovarian cancer is very complicated, is the coefficient result of polygene, will help further to understand ovarian cancer drug-resistant mechanism and detect this target.(Tatyana?A.Holzmayer，Susan?Hilsenbeck.Clinical?Correlates?of?MDR1?P-glycoprotein)Gene?Expression?in?Ovarian?and?Small-Cell?Lung?Carcinomas.JNCI?J?Natl?Cancer?Inst(1992)84(19)：1486-1491.Maria?Kavallaris，Jennifer?A.Leary，Julie?A.et，al.MDR1?and?multidrug?resistance-associated?protein(MRP)gene?expression?in?epithelial?ovarian?tumors.Cancer?letters，volume102，issues1-2，19?April?1996：7-16.)

Utilize fluorescent probe that above-mentioned eight kinds of marks are detected, can more comprehensively be familiar with the mechanism of ovarian cancer, analyze more accurately patient's the state of an illness, understand the genesis of ovarian cancer in clinical application.By molecule parting, the prognosis of assessment ovarian cancer patients is used for instructing the individualized treatment the establishment of the project, selects suitable medicine, thereby reduces mortality ratio, and the Economy type medicine cost reaches the purpose of optimizing treatment effect.

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

Preferred embodiment according to the present invention retrieves respectively by NCBI Mapview database the clone that all contain target gene, respectively these clones is screened, and selects optimum clone.

Preferred embodiment according to the present invention, this detection agent comprises the BRCA1 gene probe: GSP BRCA1

GSP BRCA1 comprises 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; The second probe comprises beyond BRCA1 gene and 5 ' end the nucleotide sequence of 102Kb beyond 23Kb and 3 ' end; Described 2 probes all are positioned at the 17q21 district.

In one embodiment of the invention, the nucleotide sequence length of described 2 probes of GSP BRCA1 is always 398Kb.Probe is bought the clone bank in Invitrogen RP11 BAC.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).

Preferred embodiment according to the present invention, this detection agent comprises the PTEN gene probe: GSP PTEN

GSP PTEN comprises 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; The second probe comprise 61Kb beyond part PTEN gene 98Kb and 5 ' end and nucleotide sequence; The 3rd probe comprises part PTEN gene 13Kb and 3 ' the end nucleotide sequence of 170Kb in addition; Described 3 probes all are positioned at the 10q23 district.

In one embodiment of the invention, the nucleotide sequence length of described 3 probes of 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).

Preferred embodiment according to the present invention, this detection agent comprises the WT1 gene probe: GSP WT1

GSP WT1 comprises 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; The 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 the 3rd probe WT1 gene 3 ' end; Described 3 probes all are positioned at the 11p13 district.

In one embodiment of the invention, the nucleotide sequence length of described 3 probes of 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).

Preferred embodiment according to the present invention, this detection agent comprises the PIK3CA gene probe: GSP PIK3CA

GSP PIK3CA comprises 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; The second probe comprise 43Kb beyond PIK3CA gene and 5 ' end and and 3 ' end beyond the nucleotide sequence of 54Kb; The 3rd probe is the nucleotide sequence of 104Kb beyond PIK3CA gene 3 ' end; Described 3 probes all are positioned at the 3q26 district.

In one embodiment of the invention, the nucleotide sequence length of described 3 probes of 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).

Preferred embodiment according to the present invention, this detection agent comprises the AIB1 gene probe: GSP AIB1

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

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

In one embodiment of the invention, the nucleotide sequence length of described 2 probes of GSP AIB1 is always 335Kb.Probe is bought the clone bank in Invitrogen RP11 BAC.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).

Preferred embodiment according to the present invention, this detection agent comprises the AURKA gene probe: GSP AURKA

GSP AURKA comprises 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; The 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 the 3rd probe AURKA gene 3 ' end; Described 3 probes all are positioned at the 20q13 district.

In one embodiment of the invention, the nucleotide sequence length of described 3 probes of 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).

Preferred embodiment according to the present invention, this detection agent comprises the ZNF217 gene probe: GSP ZNF217

GSP ZNF217 comprises 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; The second probe comprises beyond ZNF217 gene and 5 ' end the nucleotide sequence of 125Kb beyond 54Kb and 3 ' end; Described 2 probes all are positioned at the 20q13 district.

In one embodiment of the invention, the nucleotide sequence length of described 2 probes of GSP ZNF217 is always 337Kb.Probe is bought the clone bank in Invitrogen RP11 BAC.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).

Preferred embodiment according to the present invention, this detection agent comprises the MDR1 gene probe: GSP MDR1

GSP MDR1 comprises 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; The second probe be beyond MDR1 gene and 5 ' end beyond 51Kb and 3 ' end 3Kb nucleotide sequence; The nucleotide sequence of 171Kb beyond the 3rd probe MDR1 gene 3 ' end; Described 3 probes all are positioned at the 7q21 district.

In one embodiment of the invention, the nucleotide sequence length of described 3 probes of GSP MDR1 is always 473Kb.Probe is bought the clone bank in Invitrogen RP11 BAC.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 who uses screening to obtain prepares the said gene probe, the demonstration of fluorescence in situ hybridization test result, and hybridization signal is bright, can be used for pattern detection.

Preferred embodiment according to the present invention, in the gene probe preparation process, the cloned plasmids DNA extraction can be used commercially available plasmid extraction kit, includes but not limited to, as the Plasmid Maxi Kit of Qiagen company.

Preferred embodiment according to the present invention, in the gene probe preparation process, cloned plasmids DNA's is quantitatively the absorbancy that will measure respectively after the plasmid DNA dilution under 260nm and 280nm, calculates production concentration.

Preferred embodiment according to the present invention, the mark of gene probe can adopt method of the prior art with corresponding fluorescein-labelled to 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 test kit, the Nick Translation Kit of preferred abbott and/or Roche company.

Preferred embodiment according to the present invention, the fluorescein of Probe labelling can be selected fluorescence dye known in the art, as: Alexa

488, Alexa

555, pacific

FITC, DEAC, Rhodamine etc., the different probe in the same gene probe groups, as the BRCA1 gene probe: the first probe and the second probe in GSP BRCA1, use same fluorescence dye to carry out mark in principle.

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

Preferred embodiment according to the present invention, when carrying out the single index detection, in the fluorescence system, preferred isothiocyanic acid (green) or rhodamine (redness) carry out mark.

Preferred embodiment according to the present invention for gene probe, when with the rhodamine mark, under normal circumstances, should show 2 danger signals (2R) under fluorescent microscope; When homozygous mutant gene occurring, do not show danger signal (0R) under fluorescent microscope; When gene amplification occurring, a plurality of danger signals of demonstration under fluorescent microscope (＞2R).

Another object of the present invention is to utilize 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 adopted following technical scheme:

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

(2) hybridization: preparing hybrid liquid, mainly comprise fluorescence labeling probe mixture and hybridization buffer, carry out the hybridization of 8～16 hours under suitable temp, then wash away not in conjunction with upper and probe non-specific binding with suitable washing lotion; 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 the invention of above technical scheme comprises: 1) hybridization buffer, fluorescence labeling probe mixture, unlabelled competitive DNA, DAPI counterstain, and 2) separate and concentrate the 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 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 made up according to testing goal, after the different fluorescence dyes of mark, be used for the ovarian cancer molecular marker and detect respectively; 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, 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:

Detect when (1) having realized same sample is reached 4 kinds of molecular markers, be conducive to improve the early stage of ovarian cancer

Recall rate is carried out the ovarian cancer molecule parting more accurately;

(2) carry out signal-count and good reproducibility accurately and rapidly as a result;

(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 realize the application in fields such as oncobiology, cytogeneticss, help each molecular marker of comprehensive evaluation, understand contacting of itself and ovarian cancer genesis etc., auxiliary clinical targeted therapy medication and treatment plan are selected.

Description of drawings

Fig. 1 shows GSP BRCA1 probe groups site mode chart (red wire frame probe position).

Fig. 2 shows GSP PTEN probe groups site mode chart (red wire frame probe position).

Fig. 3 shows GSPWT1 probe groups site mode chart (red wire frame probe position).

Fig. 4 shows GSP ZNF217 probe groups site mode chart (red wire frame probe position).

Fig. 5 shows GSP PIK3CA probe groups site mode chart (red wire frame probe position).

Fig. 6 shows GSP AURKA probe groups site mode chart (red wire frame probe position).

Fig. 7 shows GSP AIB1 probe groups site mode chart (red wire frame probe position).

Fig. 8 shows GSP MDR1 probe groups site mode chart (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 fixedly detected result of gene probe (four looks) in paraffin-embedded tissue section of ovarian cancer tissue's formalin.

Embodiment

The following example is intended to illustrate rather than 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's culture ﹠ identification: buys corresponding clone Invitrogen RPCI11.C, get in the TB nutrient solution (chlorampenicol resistant) that 50ul adds 500ml to, shake bacterium cultivation 24～48 hours in 37 ℃ of shaking tables; Bacterium liquid uses the STS primer pair to carry out clone identification.The RP11-948G15:STS primer pair: upstream primer 5 '-CTTGCACCTATAATCCCAG-3 ' and downstream primer 5 '-AGTCTTCTTGTCTTGTGGC-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 128bps left and right has bright band as a result.The RP11-831F13STS primer pair: upstream primer 5 '-AAACATGTTCCTCCTAAGGTGCTTT-3 ' and downstream primer 5 '-ATGAAACCAGAAGTAAGTCCACCAGT-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 119bps left and right has bright band as a result.

(3) GSP BRCA1 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 ultralow copy 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/ul)=0D260 * 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water dilution to be 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 ℃ of sealings are preserved.

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

Joined rear concussion mixing, 16 ℃ of marks 12 hours, then 80 ℃ hatched 10 minutes inactivators.Get 5ul and use 2% sepharose to do electrophoresis, require to exist the band of disperse between 100-500bp.

Marked product is carried out ethanol precipitation and concentrated, add successively sodium-acetate and dehydrated alcohol by following scheme in the 1.5ml centrifuge tube, lucifuge, preparation on ice:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing is placed in-70 ℃ of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 ℃ of 13000rpm carefully remove supernatant, stir precipitation, add 70% ethanol of 1ml, 4 ℃ 13000 rev/mins centrifugal 15 minutes, carefully remove supernatant, stir precipitation, lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP BRCA1 gene probe, lucifuge ,-20 ℃ of storages.

(4) GSP BRCA1 gene probe checking: use mankind's proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus on 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: the RP11-113H4:STS primer pair: upstream primer 5 '-AGAAAGACAGACAAAAAGATGAGG-3 ' and downstream primer 5 '-GAGTTGCCAGTGTGCTTTAC-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 188bps left and right has bright band as a result.CTD-2557P6 STS primer pair: upstream primer 5 '-GTTTTTAGTGAGGGCTGGTGAAA-3 ' and downstream primer 5 '-ATTTCCCTTTGGAAAGTGCTGTT-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 341bps left and right has bright band as a result.The RP11-765C10STS primer pair: upstream primer 5 '-TGTTAAGCTTGTCTATGCTAAACAA-3 ' and downstream primer 5 '-AATTCAGTGGCTTAATCATGAATG-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 341bps left and right has bright band as a result.Other step is referring to embodiment 1.

(3) GSP PTEN 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 ultralow copy 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/ul)=OD260 * 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water dilution to be 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 ℃ of sealings are preserved.

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

Joined rear concussion mixing, 16 ℃ of marks 12 hours, then 80 ℃ hatched 10 minutes inactivators.Get 5ul and use 2% sepharose to do electrophoresis, require to exist the band of disperse between 100-500bp.

Marked product is carried out ethanol precipitation and concentrated, add successively sodium-acetate and dehydrated alcohol by following scheme in the 1.5ml centrifuge tube, lucifuge, preparation on ice:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing is placed in-70 ℃ of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 ℃ of 13000rpm carefully remove supernatant, stir precipitation, add 70% ethanol of 1ml, 4 ℃ 13000 rev/mins centrifugal 15 minutes, carefully remove supernatant, stir precipitation, lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP BRCA1 gene probe, lucifuge ,-20 ℃ of storages.

(4) GSP PTEN gene probe checking: use mankind's proper splitting lymphocyte in mid-term to drip sheet and carry out the probe checking.Comprise mid-term or interphase chromosome DNA, during fluorescence in situ hybridization, chromosomal DNA shows as discernible karyomit(e) or nucleus on 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: the RP11-1037K14:STS primer pair: upstream primer 5 '-AGCTCAAGTGGACAGATGTACAGG-3 ' and downstream primer 5 '-TGCAATTAGCACGACCATGATAC-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 323bps left and right has bright band as a result.The CTD-2643K15STS primer pair: upstream primer 5 '-GTTGTCCTTTTCAGCATTGC-3 ' and downstream primer 5 '-GCAGGGTTTCATCCTCGG-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 405bps left and right has bright band as a result.The RP11-195J14STS primer pair: upstream primer 5 '-CCTTTGCTTATGCTGCTTCC-3 ' and downstream primer 5 '-CATGCTTCATGCTTCTCTATGG-3 ', the pcr amplification condition is: 95 ℃ 5 minutes; (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 45 seconds) * 35 circulations; 72 ℃ 7 minutes.Amplified production carries out the electrophoresis checking, and the 130bps left and right has bright band as a result.Other step is referring to embodiment 1.

(3) GSP WT1 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 ultralow copy 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/ul)=OD260 * 50 (ng/ul) calculates plasmid DNA concentration.Adopt autoclaved ultrapure water dilution to be 100ng/ul, adopt the centrifuge tube packing of 1.5ml ,-20 ℃ of sealings are preserved.

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

Joined rear concussion mixing, 16 ℃ of marks 12 hours, then 80 ℃ hatched 10 minutes inactivators.Get 5ul and use 2% sepharose to do electrophoresis, require to exist the band of disperse between 100-500bp.

Marked product is carried out ethanol precipitation and concentrated, add successively sodium-acetate and dehydrated alcohol by following scheme in the 1.5ml centrifuge tube, lucifuge, preparation on ice:

Marked product 45ul

Sodium-acetate (3mol/L) 5ul

Dehydrated alcohol 125ul

Mixing is placed in-70 ℃ of refrigerators at least 2 hours, and centrifugal 30 minutes of 4 ℃ of 13000rpm carefully remove supernatant, stir precipitation, add 70% ethanol of 1ml, 4 ℃ 13000 rev/mins centrifugal 15 minutes, carefully remove supernatant, stir precipitation, lucifuge is dry.Use 1ul purified water dissolution precipitation, obtain GSP WT1 gene probe, lucifuge ,-20 ℃ of storages.

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

Annotate: 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)

Take 10 person-portions/box as example.

(1) hybridization solution preparation

The probe that mark is good is put well successively, at first dissolves probe.Use 1 μ l sterilization purified water to dissolve in the gene probe dry powder that is prepared by embodiment 1～4 method, fully mixing.Press 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, 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 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 1,2,3,4	Each 100 μ l/ pipe	4 pipes
The DAPI counterstain	400 μ l/ pipes	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)

Take 10 person-portions/box as example.

(4) hybridization solution preparation

The probe that mark is good is put well successively, at first dissolves probe.Use respectively 1 μ l sterilization purified water to dissolve in the gene probe dry powder that is prepared by embodiment 1～4 method, fully mixing.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

The component title	Specification	Quantity
			Hybridization solution 1,2	Each 100 μ l/ pipe	2 pipes
The DAPI counterstain	200 μ l/ pipes	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)

Take formalin fixedly the ovary tissue sample that buries of paraffin as example.

(1) slide pre-treatment

Slide is put into the roasting sheet of 65 ± 5 ℃ of thermostat containers 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 dehydrated alcohol 10 minutes, put into the room temperature dehydrated alcohol 10 minutes, then put into successively room temperature 100% ethanol, 90% ethanol, 70% ethanol each 3 minutes; In deionized water at room temperature standing 3 minutes, draw excessive moisture; (section was placed horizontally in container in 25 minutes to boil sheet in the deionized water of 100 ± 5 ℃, sample faces up), after drying, the Proteinase K reaction solution of putting into 37 ± 1 ℃ of preheatings digested about 8～15 minutes, in room temperature 2 * SSC, rinsing is 5 minutes, put into successively room temperature 70%, 90%, 100% gradient ethanol dehydration each 3 minutes.Dry slide; Continue crossover process.

(2) the same time variation of sample and probe

Take out hybridization solution from test kit, the concussion mixing, instantaneous centrifugal; Add the hybridization solution of 10 μ l to the hybridization zone, covered, gently press hybridization solution is evenly distributed rapidly, avoids producing bubble; Rubber cement covers along cover glass edge mounting the edge that cover glass contacts with slide glass fully; On the hot platform of 85 ± 1 ℃, sex change is 5 minutes; Slide is put into the hybridizing box of preheating, lucifuge, 37 ± 1 ℃ of overnight incubation (approximately 16 hours); Continue the post-hybridization washing step.

(3) post-hybridization washing and redying

Take out the hybridizing box of overnight incubation, carefully remove rubber cement and cover glass; Slide was put into 37 ± 1 ℃ of 2 * SSC 10 minutes; Put it into again 37 ± 1 ℃ of 0.1%NP-40/2 * SSC washing 2 minutes; 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 BX50 fluorescent microscope, observe respectively with the filter group hybridization fluorescent signal that DAPI redyes, use the CCD photographic recording.Seek under 40 * object lens, count under 100 * object lens; 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 core; The number of each probe in each cell observed in record.

(5) result is judged

Operate by the treatment process requirement, record the signal number of each gene probe.The fluorescence in situ hybridization result shows, on Metaphase Chromosome, equal 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 (referring to accompanying drawing 12).

Embodiment 8: the clinical in-service evaluation of ovarian cancer molecular marker fluorescence in situ hybridization detection test kit

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

Table 10 clinical sample case information

Table 11 ovarian cancer molecular marker detected result

Annotate: normal N; Amplification A; Disappearance D.

Detected result is explained:

1. BRCA1 disappearance prompting ovarian tumor occurs.

2. generation and the evolution of PTEN disappearance prompting ovarian tumor may be early molecule event in the ovarian cancer generation.

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

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

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

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

7. the AIB1 amplification indicates patient's poor prognosis.

8. platinum class resistance, measurable curative effect and toxic side effect are pointed out in the MDR1 amplification.

Know from detected result, after these samples being carried out the molecular marker detection, can carry out molecule parting to sample according to detected result, according to the meaning that detects index, the stage and step of ovarian cancer be can assist, simultaneously, clinical treatment formulation, medication selection and curative effect judgement are used for.

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

Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that concrete enforcement 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 (8)

1. ovarian cancer molecular marker detection kit, comprise: 1) hybridization solution, DAPI counterstain, with 2) separate and concentrate the packing box of packing these reagent bottles or pipe, wherein hybridization solution is formulated by hybridization buffer, fluorescently-labeled BRCA1 and/or PTEN and/or WT1 and/or PIK3CA and/or ZNF217 and/or AURKA and/or AIB1 and/or MDR1 gene probe and sealing DNA.

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

3. detection kit according to claim 1 is further characterized in that described detection agent comprises following probe:

(1)GSPBRCA1

Comprise 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; The second probe comprises beyond BRCA1 gene and 5 ' end the nucleotide sequence of 102Kb beyond 23Kb and 3 ' end; Described 2 probes all are positioned at the 17q21 district

(2)GSP?PTEN

Comprise the first probe, the second probe and the 3rd probe, the first probe nucleic acid sequence is the nucleotide sequence of the 487Kb beyond PTEN gene 5 ' end; The second probe comprise 61Kb beyond part PTEN gene 98Kb and 5 ' end and nucleotide sequence; The 3rd probe comprises part PTEN gene 13Kb and 3 ' the end nucleotide sequence of 170Kb in addition; Described 3 probes all are positioned at the 10q23 district

(3)GSPWT1

Comprise the first probe, the second probe and the 3rd probe, the first probe nucleic acid sequence is the nucleotide sequence of the 182Kb beyond WT1 gene 5 ' end; The 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 the 3rd probe WT1 gene 3 ' end; Described 3 probes all are positioned at the 11p13 district

(4)GSP?PIK3CA

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

(5)GSP?AIB1

Comprise the first probe and the second probe, the first probe nucleic acid sequence comprises AIB1 gene 126Kb and 5 ' the end nucleotide sequence of 36Kb in addition; The second probe comprises AIB1 gene 47Kb and 3 ' the end nucleotide sequence of 145Kb in addition; Described 2 probes all are positioned at the 20q13 district

(6)GSP?AURKA

Comprise the first probe, the second probe and the 3rd probe, the first probe nucleic acid sequence is the nucleotide sequence of the 190Kb beyond AURKA gene 5 ' end; The 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 the 3rd probe AURKA gene 3 ' end; Described 3 probes all are positioned at the 20q13 district

(7)GSP?ZNF217

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

(8)GSP?MDR1

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

Above-mentioned probe is selected different fluorochrome labels.

4. detection kit according to claim 1, be further characterized in that described gene probe prepares template with clone's Insert Fragment of inclusion test target gene and peripheral nucleotide sequence as probe, adopts random primer or nick-translation method to carry out fluorescent mark.

5. detection kit according to claim 1 is further characterized in that gene probe is preferably as follows clone's combination:

(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。

6. according to claim 1 test kit, be further characterized in that described gene probe group can arbitrary combination.

7. according to claim 1 test kit, be further characterized in that described test kit is detecting ovarian cancer, or the purposes in other noumenal tumour (cervical cancer, bladder cancer, the esophageal carcinoma, lung cancer, mammary cancer, skin carcinoma, kidney etc.) and hemopathy (lymphoma, leukemia etc.).

8. according to claim 1 test kit, be further characterized in that the sample that detects is tissue samples or cell sample.

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