CN109234392A - Purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product - Google Patents

Abstract

The invention discloses purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product, the biomarker is GPR56 and/or NWD2 gene and its expression product.The present invention has found 2 kinds of genes relevant to breast cancer by transcript profile sequencing, and studies its application prospect in breast cancer early detection；For the cause of disease pathogenesis for further studying breast cancer, the drug target for exploring breast cancer early prevention and treatment provides new direction.

Description

Purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product

Technical field

The present invention relates to technical field of biomedical detection, and in particular to marker preparation breast cancer detection, diagnosis or Purposes in Prognosis scoveillance product.

Background technique

Breast cancer is that a kind of systemic disease, its occurrence and development are one and are related to multifactor, too many levels complex process, The inactivation etc. of activation and tumor suppressor gene including oncogene.Therefore, gene mutation rises in the generation, development process of breast cancer Very important effect.

Breast cancer is a multifactor hereditary variability disease, is only due to caused by single-gene defect less than 10%. It is more and more to be found with breast cancer related gene with the development of high-throughput gene technology, potential heredity on these genes Variation (mononucleotide polymorphic and copy number variation) may cause the difference of breast cancer medicines therapeutic effect.Due to hereditary variation May be affected in the presence of the metabolic pathway and pharmaceutically-active target gene for making anti-tumor drug, so affect the treatment and Prognosis.

RCA1 and BRCA2 is the identified earliest and most common Familial Occurrence breast cancer characteristic gene.With correlation The application of new technology, existing lots of genes are reported related to breast cancer successively.These genes can substantially be divided into " high risk " With " low to moderate risk " breast cancer susceptibility gene, wherein " high risk " tumor susceptibility gene includes: BRCA1 and BRCA2 (in young man Middle disease incidence is much higher)；And PTEN, TP53, CHEK2, TGF β 1, PALB2 and ATM belong to " moderate risk " breast cancer susceptibility base Cause.Studies have shown that being diagnosed as in the women of patient with breast cancer before 40 years old, about 1% is found to carry the mutation of p53 gene； It was diagnosed as in the women of patient with breast cancer before 30 years old, up to 4% carries the mutation of p53 gene.Carry PTEN homologous deletion The women of mutation, the risk to suffer from breast cancer are 25-50%, this is more common in young woman.The female of PALB2 gene mutation Property, it suffers from breast cancer and increases with the risk of cancer of pancreas.In addition there are also 90 common " low risk " mutation.These above-mentioned variations are only Explain the risk of 37% familial breast cancer.Still there is the familial breast cancer onset risk close to 2/3rds to can not find pair The Disease-causing gene answered.

There are two familial breast cancer refers in a family or the above member with genetic connection suffers from breast cancer, and accounts for cream The 5-7% of gland cancer totality.Compared with sporadic breast cancer, the disease incidence of familial breast cancer is higher, and young (< 40 years old) is early Hair property patient, prognosis is worse, and survival rate is lower.Large-scale epidemiological study shows for having a first degree relative illness Body, the probability of pathogenesis of breast carcinoma are 5.5%；There are two the individual of first degree relative illness, disease incidence 13.3%.Evans Et al. further retrospective analysis show compared with normal control population, the family's individual for thering is first degree relative to suffer from breast cancer, hair Sick rate increases 2-4 times.Familial Occurrence is relatively conventional in young early breast cancer group.Race's property mammary gland is found as it can be seen that exploring Cancer associated gene will provide new theoretical foundation and clinical guidance for the early diagnosis, treatment and prognosis of familial breast cancer.

Summary of the invention

To solve the above-mentioned problems, it is an object of the invention to find the biological marker that can be used in breast cancer early detection Object simultaneously provides its related application.

It is described present invention firstly provides purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product Marker is GPR56 and/or NWD2 gene and its expression product.

Further, the marker is that GPR56 and/or NWD2 gene is lowered in breast carcinoma.

Further, product mentioned above includes: miscellaneous by RT-PCR, Western method, immune detection, original position It hands over, chip or kit detect the expression of said gene and its expression product with the product of Diagnosis of Breast cancer.

Further, the product with RT-PCR Diagnosis of Breast cancer includes at least a pair of of specific amplified GPR56 and/or NWD2 The primer of gene；The product with immune detection Diagnosis of Breast cancer includes: in conjunction with GPR56, and/or NWD2 protein-specific Antibody；The product in situ hybridization Diagnosis of Breast cancer includes: the nucleic acid array hybridizing with GPR56 and/or NWD2 gene Probe；The product with chip Diagnosis of Breast cancer includes: protein chip and genetic chip；Wherein, protein chip include with The antibody that GPR56 and/or NWD2 protein-specific combines, genetic chip includes the nucleic acid sequence with GPR56 and/or NWD2 gene The probe of hybridization.

With the probe of the nucleic acid array hybridizing of GPR56 and/or NWD2 gene can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives.There is no limit as long as complete specific hybrid and purpose nucleotide sequence spy for the length of the probe The opposite sex combines, and any length is ok.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally, institute The length for stating probe can be grown to 60,80,100,150,300 base-pairs or longer or even whole genes.Due to different probes Length has different influences to hybridization efficiency, signal specificity, and the length of the probe is typically at least 14 base-pairs, longest 30 base-pairs are usually no more than, the length complementary with purpose nucleotide sequence is best with 15-25 base-pair.The probe is certainly Body complementary series is most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.

Further, the specific antibody of the GPR56 and/or NWD2 albumen includes monoclonal antibody, polyclonal antibody.Institute The specific antibody for stating GPR56 and/or NWD2 albumen includes any segment or modification of complete antibody molecule, antibody, for example, Chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc..As long as the segment can retain and the combination of GPR56 and/or NWD2 albumen Ability.Well known to a person skilled in the art and the present invention can be used when preparation for the antibody of protein level Any method prepares the antibody.

Preferably, the product includes chip, kit.

Further, the solid phase carrier includes inorganic carrier and organic carrier, and the inorganic carrier includes but is not limited to have Silicon carrier, glass carrier, ceramic monolith etc.；The organic carrier includes polypropylene film, nylon membrane etc..

Further, the kit includes gene detecting kit and protein immunization detection kit；The genetic test Kit includes the reagent for detecting GPR56 and/or NWD2 gene transcription level；The protein immunization detection kit includes The specific antibody of GPR56 and/or NWD2 albumen.

Preferably, the kit is breast cancer detection kit, and it includes detection GPR56 and/or NWD2 gene expressions Horizontal tool, and how to use the explanation of the kit.

The tool includes the primer pair or probe for detecting GPR56 and/or NWD2 gene.The GPR56 primer pair With nucleotide sequence shown in SEQ IDNO:1 and SEQ ID NO:2；The NWD2 primer pair has SEQ IDNO:3 and SEQ Nucleotide sequence shown in ID NO:4.

Preferably, the kit further includes 10 × Buffer, dNTP, MgCl₂, Taq enzyme and SYBR Green fluorescence dye Material.

Further, the present invention also provides GPR56 the and/or NWD2 genes and its expression product treats in preparation Application in the drug of breast cancer.

Drug of the invention can also mention significantly with the drug combination of other treatment breast cancer, a variety of Drug combinations The success rate of height treatment.

Drug of the invention further includes pharmaceutically acceptable carrier, and this kind of carrier includes (but being not limited to): diluent, Excipient such as lactose, sodium chloride, glucose, urea, starch, water etc., filler such as starch, sucrose etc.；Adhesive such as simple syrup, Glucose solution, starch solution, cellulose derivative, alginates, gelatin and polyvinylpyrrolidone；Wetting agent such as glycerol；It collapses Solve agent such as dried starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate；Sorbefacient is quaternized Close object, lauryl sodium sulfate etc.；Surfactant for example polyoxyethylene sorbitan aliphatic ester, lauryl sodium sulfate, Glyceryl monostearate, hexadecanol etc.；Humectant such as glycerol, starch etc.；Absorption carrier such as starch, lactose, bentonite, silicon Glue, kaolin and soap clay etc.；Lubricant such as talcum powder, calcium stearate and magnesium, polyethylene glycol, boric acid powder etc..

Drug of the invention can be used different additives and be prepared, for example, stabilizer, fungicide, buffer, etc. Penetration enhancer, chelating agent, pH controlling agent and surfactant.

Stabilizer includes Human serum proteins, l-amino acid, sugar and cellulose derivative.L-amino acid can also include sweet Any one in propylhomoserin, cysteine and glutamic acid.Carbohydrate includes monosaccharide, such as glucose, mannose, galactolipin, fructose Deng；Sugar alcohol, such as mannitol, inositol, xylitol etc.；Disaccharides, such as sucrose, maltose, lactose etc.；Polysaccharide, such as Portugal Glycan, hydroxypropul starch, vulcanization chondroitin, hyaluronic acid etc. and their derivative.Cellulose derivative includes Methyl cellulose Element, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose and sodium cellulose glycolate.

Surfactant includes ion or nonionic surfactant, such as polyethylene glycol oxide Arrcostab, sorbitan list Acyl ester, fatty glyceride.

Additive buffer may include boric acid, phosphoric acid, acetic acid, citric acid, glutamic acid and corresponding salt (their alkali gold Category or alkaline rare earth metal salt, such as sodium salt, sylvite, calcium salt and magnesium salts).Isotonic agent includes potassium chloride, sodium chloride, sugar and sweet Oil.Chelating agent includes sodium ethylene diamine tetracetate and citric acid.

Drug of the invention may also include pharmaceutically acceptable coating material, fast decoupled coating Material, coloring agent, enteric polymer, plasticizer, water-soluble polymer, insoluble polymer, dyestuff, pigment, other collapse Powder.Common fast decoupled coating material includes OPADRY；Enteric polymer includes methylacrylic acid polymer, phosphorus hydroxypropyl first Base cellulose phthalate, hypromellose acetic acid esters, hypromellose succinate, hydroxyl first ethyl cellulose Element, cellulose acetophthalate；Plasticizer includes polyethylene glycol (PEG), propylene glycol etc..

The unit dosage forms of drug of the present invention can make diversified forms, and representative dosage form includes solid dosage forms such as tablet, ball Agent, pulvis, dry powder doses, particle, capsule etc.；Liquid forms such as solution, suspension, emulsion, syrup, elixir etc..

Drug of the present invention can give receptor by any approach, as long as can reach destination organization, can pass through mouth Clothes or non-oral number of ways, such as oral administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intradermal are given intranasal administration Medicine, feeding drug into pulmones, drop rectum with drug, intravenous administration.

Beneficial effect

The present invention has found 2 kinds of genes relevant to breast cancer by transcript profile sequencing, and studies it in breast cancer morning Application prospect in phase detection；For the cause of disease pathogenesis for further studying breast cancer, the drug of breast cancer early prevention and treatment is explored Target spot provides new direction.

Detailed description of the invention

Fig. 1 is expression of the GPR56 and NWD2 gene of the present invention in the breast cancer case group in RT-PCR.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment Used in the conventional means that are well known to those skilled in the art of technological means.

Test method without specific conditions in embodiment, usually conventional method in that art.

In the context of the present invention, " GPR56 gene " includes any function etc. of GPR56 gene and GPR56 gene The polynucleotides of jljl.GPR56 gene includes and GPR56 gene in the public GenBank GeneBank in the current world (NC_000016.10) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein.

In the context of the present invention, " NWD2 gene " includes any functional equivalent of NWD2 gene and NWD2 gene Polynucleotides.NWD2 gene includes and NWD2 gene (NC_ in the public GenBank GeneBank in the current world 000004.12) DNA sequence dna has 70% or more homology, and encodes the DNA sequence dna of identical function protein.

The different Familial Occurrence breast cancer sample collections of embodiment 1 and clinical data acquisition

1, patient with breast cancer's family is included in standard

1) there are 1 or more patient with breast cancer, and at least 1 in first degree relative in addition to propositus in a family Example meets one of following condition: when morbidity < 40 years old；There is simultaneously or successively BILATERAL BREAST CANCER；There is simultaneously or successively non-mammary gland Malignant tumour；

2) preoperative not receive any associated treatment, such as radiotherapy, chemotherapy, endocrine therapy and targeted therapy；

3) meet the international clinical stages I-IV phase and without taboo person, row related surgical curer (including breast tumor excision Art is for mammary cancer surgery, radical mastectomy, modified skinsuture and Breast reservation, in conjunction with or do not combine axillary lymph node dissection Art).

2, patient with breast cancer's family exclusion criteria

1) preoperative row auxiliary puts/chemotherapeutic treatment；

2) clinical symptoms, sign, auxiliary examination are combined, or guide lower breast lump needle aspiration biopsy to examine through B ultrasound Break as benign lesion, nothing clearly operation pointer person；

3) patient of operation materials cannot be carried out there are other surgical contraindications；

4) after operation excision, cancerous swelling is too small (maximum gauge < 1cm), and there are pathological diagnosis difficulty；

5) it is associated with the patient of other rheumatism immunological diseases such as rheumatoid arthritis, rheumatic fever；

6) patient of other serious systemic diseases is suffered from；

7) epidemiologic data, clinical data and the imperfect person of image data；

8) informed consent person is not obtained.

3, sample collection

Obtain the peripheral blood and 5 relatively normal peripheral blood samples of 5 patient with breast cancers.All patients are preoperative not to be connect By chemotherapy or radiotherapy, the preoperative informed consent for having obtained patient, and audited by Ethics Committee.

Sample process: EDTA anticoagulated whole blood is mixed in 1:1 ratio with Trizol, mixes well and is placed on 1.8mL cell In cryopreservation tube, the refrigerator preservation that 30s is placed on -80 DEG C is cooled down rapidly in liquid nitrogen.

2 peripheral blood RNA of embodiment is extracted

1, homogenized

Fresh blood is taken, 3 times of volume erythrocyte cracked liquids are added, 10min, 10000rpm centrifugation are placed at room temperature for after mixing 1min.Supernatant is abandoned, leukocyte cell pellet is collected.1ml Trizol is added in the leukocyte cell pellet of every 100-200 μ L blood collection.

2, it is layered

After Trizol is added in sample, it is placed at room temperature for 5min, cracks sample sufficiently.

200 μ L chloroforms are added in every 1mL Trizol, and acutely concussion, which is placed at room temperature for 3-5min, after mixing makes its natural split-phase.

3, RNA precipitate

4 DEG C of 12000rpm are centrifuged 10-15min.Sample can be divided into 3 layers: yellow organic phase, middle layer and colourless aqueous phase, RNA Mainly in water phase, water phase (can usually draw 550-600 μ L) is transferred in new EP pipe.Note: water phase is carefully drawn, necessarily not Intermediate interface is drawn, otherwise will lead to has DNA pollution in RNA sample.

The isopropanol being pre-chilled in advance in equal volume is added in supernatant, is placed at room temperature for 10-20min.4 DEG C of 12000rpm centrifugations 10min abandons supernatant, and RNA precipitate is in tube bottom.

4, RNA is rinsed

1ml75% ethyl alcohol (being prepared with RNase-free water) is added in RNA precipitate, mildly vibrates centrifuge tube, suspend precipitating. Do not have 1mL Trizol that 1mL75% ethyl alcohol is added.

4 DEG C of 5000-8000rpm are centrifuged 1-2min, abandon supernatant；Can of short duration rapid centrifugation, with pipettor carefully inhale abandon supernatant, It is careful not to inhale and abandons precipitating.It is placed at room temperature for 1-2min and dries precipitating.

5, RNA dissolves

50-100 μ L RNase-free water is added in precipitating, flicks tube wall, sufficiently to dissolve RNA, -80 DEG C of preservations.

6, RNA integrality and purity detecting

Integrality: RNA can use plain agar sugar gel electrophoresis (deposition condition: 1.2% glue；0.5 × TBE electrophoretic buffer； 150v, 15min) detection integrality.Maximum rRNA brightness should be 1.5-2.0 times of time big rRNA brightness in RNA sample, otherwise table Show the degradation of RNA sample.There is disperse sheet or band disappearance shows that sample is seriously degraded.

Purity: OD260/OD280 ratio is the index for measuring protein contamination degree in RNA sample.The RNA sample of high quality Product, OD260/OD280 ratio (10mM Tris, ph7.5) is 2.0 or so.OD260/OD280 reading is by measuring solution used PH value influences.The same RNA sample, it is assumed that the OD260/OD280 reading determined in 10mM Tris, ph7.5 solution exists Between 1.8-2.1, measure in aqueous solution reading may between 1.5-1.9, but this not represent RNA impure.

Concentration: removing a certain amount of RNA extract, dilutes n times with RNase-free water, will be divided light with RNase-free water Degree meter zeroing, takes dilution to carry out OD260 measurement, according to the calculating of following formula progress RNA concentration: final concentration (ng/ μ L)= OD260 × n (extension rate) × 40.

The building of 3 RNA-seq sequencing library of embodiment and quality inspection

1, data quality accessment

The building and sequencing of cDNA library, commission Beijing Nuo Hezhi source Science and Technology Co., Ltd. completes.All samples Sequencing data is checked by sequencing error rate inspection, G/C content distribution and initial data filtering, obtains what subsequent analysis used Clean reads, data summarization.It has been generally acknowledged that RNA-seq data carry out the analysis of gene differential expression between different libraries, library is total Read number is at least 10M, and data entirety G/C content is maintained at 40%-60%, and Q30 is then reasonable 80% or more.This sequencing Clean bases accounts for 7.2G or more in data acquired, and Q2 base, which accounts for 95.18% or more, Q3 base and accounts for 89.15% or more, GC, to be contained Amount keeps stablizing between sample, between 54.29%-56.80%, illustrates that whole sequencing quality is good, meets downstream analysis matter Amount demand.

2, comparison result is analyzed

The clean reads of all samples STAR software is compared to reference genome sequence, average each sample Comparison rate reaches 92.51% or more, Uniquely mapping rate and refers to the reads number for comparing and arriving the single position of genome The percentage of total cleanreads number is accounted for, only Uniquely mapped reads could be used for expression quantity and count, in this research Average comparison rate with reference to genome single site is 76.956%.

3, quantitative result is analyzed

3.1 quantitative result explanations

Each sample mean output 6Gb data.Sequencing reads is compared to reference genome and after reconstructing transcript, The expression of gene all in 10 samples is calculated according to FPKM expression quantity.Then, we are with bowtie by reads It compares on gene, each sample mean detects 11552 genes.

It carries out gene level respectively to each sample or transcript degree is quantitative, remerge the expression square for obtaining all samples Battle array, first is classified as gene or transcript ID, remaining is classified as the original readcount value of each sample.

The distribution of 3.2 expressions

The gene expression values of RNA-seq are usually indicated with RPKM or FPKM.RPKM is used for single-ended sequencing, and FPKM is used for both-end Sequencing is first corrected sequencing depth, then is corrected to the length of gene or transcript.

3.3 correlation analysis

We require R2 between biology repeat samples to be at least greater than 0.8, otherwise need to make sample suitable explanation, Or re-start experiment.According to the expression value (RPKM or FPKM) of all genes of each sample, sample in calculating group and between group Relative coefficient is depicted as thermal map, can intuitively show differences between samples and the interior sample repetition situation of group between group.Correlation system between sample Number is higher, and expression pattern is more close.

4, difference results are analyzed

The input data of gene difference analysis is original readcount data obtained in gene quantification.This research uses DESeq2 software compares and analyzes normal group and illness group, screens normal group and illness group.1500 are finally filtered out altogether Difference expression gene screens cream by clustering, the enrichment of GO function and the enrichment analysis of KEGG signal path function, inventor Gland cancer marker is GPR56 and NWD2 gene, these genes are down-regulated gene in breast cancer.

Each marker gene expression in 4 patient with breast cancer DNA of embodiment

1. experimental material

It is obtained from the breast cancer for receiving 30 patients of mammary cancer surgery in Shenzhen Second Academy in January, 2013 in December, 2017 Fresh sample tissue, 20 relatively normal fresh samples.All patients are preoperative not to receive chemotherapy or radiotherapy, preoperative to have obtained The informed consent of patient, and audited by Ethics Committee.

Sample process: all sample standard deviations confirm that -80 DEG C of low temperature refrigerators of number postposition are saved through pathological examination.

2. the extraction of sample rna

Referring to embodiment 2.

3. reverse transcription synthesizes cDNA

3.1 first chain cDNA synthetic agent box (RevertAid Premium Reverse Transcriptase) (Thermo Scientific^TMEP0733)

The synthesis of 3.2 the first chains of cDNA

(1) following reagent is added in the nuclease-free PCR pipe of ice bath:

(2) 3~5s is centrifuged after mixing gently, for reaction mixture after 65 DEG C of warm bath 5min, ice bath 2min is then centrifuged for 3 ~5s.

(3) by test tube ice bath, following reagent is added:

4.0μL 5*RT Buffer

0.5μL Thermo Scientific RiboLock RNase Inhibitor(20U)

1.0μL RevertAid Premium Reverse Transcriptase(200U)

(4) 3~5s is centrifuged after mixing gently

(5) reverse transcription reaction is carried out according to following condition in PCR instrument

1. 25 DEG C of incubation 10min

2. cDNA synthesizes 50 DEG C of 30min

3. terminating 85 DEG C of 5min of reaction, after processing, it is placed in and places on ice

(6) -20 DEG C of above-mentioned solution are saved.

4.Real-Time PCR

Design of primers

Using online primer-design software Primer-BLAST design primer, each gene is utilized in ncbi database MRNA sequence design primer, interior participation in the election GAPDH are synthesized by Shanghai Sangon Biotech Company after design of primers.Specific primer sequence is as follows:

1 Real-Time PCR primer sequence of table

Operating process is as follows:

(1) reaction system: Power is usedGreen PCR Master Mix is expanded, and experimental implementation is by production Product specification carries out.Amplification program are as follows: 95 DEG C of 3min pre-reactions, the amplification for carrying out 45 circulations (95 DEG C of 3s, 60 DEG C of 30s) are anti- It answers.

2 Real-Time PCR reaction system of table

Component	Additional amount
		2×mix	10μL
Upstream primer (10 μM)	0.4μL
		Downstream primer (10 μM)	0.4μL
Template	2μL
		Sterile purified water is added	To 20 μ L

(2) sample Real-Time PCR is detected

2 μ L will be taken to make template after cDNA10 times of each sample dilution, respectively with target gene primer and reference gene primer into Row amplification.Simultaneously in 60-95 DEG C of progress solubility curve analysis.

5. experimental result

Real-time quantitative PCR amplification curve inflection point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher.Sample amplified production is molten Solution curve be all it is unimodal, illustrate that amplified production only has one, be specific amplification.According to the relative quantification formula of qRT-PCR, than Expression of more each gene in breast cancer case group and control group.As a result such as Fig. 1 is shown: qRT-PCR stable amplification result, Expression of the GPR56 and NWD2 gene in breast cancer case group is respectively the 0.46 of control tissue, 0.27 times.The result Differential gene GPR56 and NWD2 that transcript profile is sequenced is also demonstrated in breast cancer low expression as a result, quickly early for gene Phase screening breast cancer provides possibility.

The production of 5 detection kit of embodiment

Based on the primer that embodiment 4 obtains, the kit of the present invention for breast cancer detection, the kit are assembled Primer pair including specific amplified GPR56 and/or NWD2 gene is as shown in table 1；Specifically:

1, kit one includes the primer pair of specific amplification GPR56: SEQ ID NO:1 and SEQ ID NO:2；

2, kit two includes the primer pair of specific amplification NWD2: SEQ ID NO:3 and SEQ ID NO:4；

3, kit three includes the primer pair of specific amplification GPR56 and NWD2: SEQ ID NO:1 and SEQ ID NO:2； SEQ ID NO:3 and SEQ ID NO:4；

Kit of the invention further includes the primer pair of specific amplified house-keeping gene (GAPDH): SEQ ID NO:5 and SEQ Shown in ID NO:6；It further include SYBR Green polymerase chain reaction system, as PCR buffer, SYBR Green fluorescence contaminate Material, dNTPs.The ingredient of the PCR buffer is 25mM KCl, 2.5mM MgCl₂, 200mM (NH₄)₂SO₄.By dense to primer The optimization of degree and annealing temperature, final to determine that reaction system is as shown in table 3:

3 PCR reaction system of table

Component	Additional amount
		SYBR Green polymerase chain reaction system	12.5μL
Upstream primer (10 μM)	0.5μL
		Downstream primer (10 μM)	0.5μL
Template cDNA	2.0μL
		Sterile purified water is added	To 25 μ L

Optimum reaction condition are as follows: 95 DEG C of initial denaturation 5min, (95 DEG C of denaturation 15sec, 60 DEG C of annealing 45sec, 72 DEG C extend 35sec) × 40 circulation, 72 DEG C of extension 15min.

For convenience of use, kit also may include control: the normal cDNA sample of said gene one or more.

Subject's biological sample is taken, is extracted from biological sample using conventional method (or using specific kit) RNA is carried out PCR with condition according to optimal reaction system and reacted, made using cDNA normal in kit using seminal plasma fructose detection kit For the control cDNA in Real-Time PCR quantitative detection, GPR56 and/or NWD2 gene in subject's biological sample is detected The relatively normal cDNA of expression quantity expression quantity variation.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Beijing is caused into biomedical Science and Technology Ltd.

<120>purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product

<130> P18093

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> DNA sequence

<400> 1

gctacagccg aagaatgtga c 21

<210> 2

<211> 21

<212> DNA

<213> DNA sequence

<400> 2

agcaggatgt ttgggtttct c 21

<210> 3

<211> 20

<212> DNA

<213> DNA sequence

<400> 3

ccagtttgtg gtctcgctct 20

<210> 4

<211> 20

<212> DNA

<213> DNA sequence

<400> 4

gtcattccca ccacgaaggt 20

<210> 5

<211> 20

<212> DNA

<213> DNA sequence

<400> 5

aatgggcagc cgttaggaaa 20

<210> 6

<211> 20

<212> DNA

<213> DNA sequence

<400> 6

gcgcccaata cgaccaaatc 20

Claims (10)

1. purposes of the marker in preparation breast cancer detection, diagnosis or Prognosis scoveillance product, which is characterized in that the marker For GPR56 and/or NWD2 gene and its expression product.

2. purposes as described in claim 1, which is characterized in that the gene is lowered in breast carcinoma.

3. purposes as claimed in claim 1 or 2, which is characterized in that the product includes: by RT-PCR, the side Western The expression of gene described in method, immune detection, in situ hybridization, chip or kit detection claim 1 and its expression product With the product of Diagnosis of Breast cancer.

4. a kind of breast cancer detection kit, which is characterized in that the kit includes detection GPR56, and/or NWD2 gene table Up to horizontal tool, and how to use the explanation of the kit.

5. kit as claimed in claim 4, which is characterized in that the tool includes for detecting GPR56 and/or NWD2 base The primer pair or probe of cause.

6. kit as claimed in claim 5, which is characterized in that the GPR56 primer pair has SEQ IDNO:1 and SEQ Nucleotide sequence shown in ID NO:2；The NWD2 primer pair has nucleotide shown in SEQ IDNO:3 and SEQ ID NO:4 Sequence.

7. kit as claimed in claim 4, which is characterized in that the kit further include 10 × Buffer, dNTP, MgCl₂, Taq enzyme and SYBR Green fluorescent dye.

The application of 8.GPR56 and/or NWD2 gene and its expression product in the drug of preparation treatment breast cancer.

9. drug as claimed in claim 8, which is characterized in that the drug further includes the drug of other treatment breast cancer.

10. drug as claimed in claim 8 or 9, which is characterized in that the drug further includes pharmaceutically acceptable carrier.