CN106868128A

CN106868128A - The biomarker of one group of auxiliary diagnosis breast cancer and its application

Info

Publication number: CN106868128A
Application number: CN201710093427.4A
Authority: CN
Inventors: 何劲松; 陈伟财; 罗雪莹; 潘悦; 刘宝儿; 李峰
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2017-02-21
Filing date: 2017-02-21
Publication date: 2017-06-20
Anticipated expiration: 2037-02-21
Also published as: CN106868128B

Abstract

The invention discloses one group of biomarker of auxiliary diagnosis breast cancer, the biomarker is SNP site rs114337127, rs13306048, the combination of rs139477122, rs140787599, rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497.Development and application that the present invention passes through SNP gene type diagnostics reagent and diagnostic kit, may be such that the diagnosis of breast cancer is more convenient and easy, for clinician quick and precisely grasps conditions of patients, for clinical therapeutic efficacy evaluation lays the foundation, and to find that the new small molecule drug targets with potential therapeutic value provide help.

Description

The biomarker of one group of auxiliary diagnosis breast cancer and its application

Technical field

The present invention relates to field of biomedicine technology, and in particular to the biomarker of one group of auxiliary diagnosis breast cancer and its Using.

Background technology

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, Including the activation of oncogene and the inactivation of tumor suppressor gene etc..Therefore, gene mutation rises in the generation, evolution of breast cancer Very important effect.

Breast cancer is a multifactor hereditary variability disease, only less than 10% because single-gene defect causes. It is more and more to be found with breast cancer related gene with the development of high flux 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 In the presence of making the metabolic pathway of antineoplastic and pharmaceutically-active target gene to be affected, so affect the treatment and Prognosis.

SNP (singlenucleotidepolymorphism, SNP, i.e. SNP) is 1996 by the U.S. The molecule genetic marker that the human genome research center scholar Lander of the Massachusetts Institute of Technology is proposed, is primarily referred to as gene DNA sequence polymorphism in group level as caused by the variation of single nucleotide acid.The polymorphism that SNP shows relates only to single The variation of base, performance is that have conversion, transversion, insertion and missing etc..SNP is third generation genetic marker, human body Many phenotypic differences, all may be relevant with SNP to neurological susceptibility of medicine or disease etc..It is pre- for different parting breast cancer at present Afterwards, the predictive research of curative effect is concentrated mainly on SNP levels.

SNP assigns the individual differential responses to environmental exposure, drug therapy etc., so that different phenotypes are produced, therefore SNP It is probably the important hereditary basis for causing individual disease development difference.Diagnosed the illness using the SNP spectrums of disease-susceptible humans, had Quickly, sensitive, accurate the features such as, thus have a extensive future.In recent years, the generation development for being diagnosed the illness using SNP has been turned into The study hotspot of clinical and researcher.

However, there is presently no SNP to be applied to the report of breast cancer diagnosis, if the SNP of breast cancer susceptibility can be filtered out As biomarker, and corresponding diagnostic kit is developed, will effectively promote the present situation of China's early diagnosing mammary cancer, And for its drug screening, evaluating drug effect and targeted therapy open up new approach.

The content of the invention

The purpose of the present invention is directed to above-mentioned technical problem, proposes one group of biomarker of auxiliary diagnosis breast cancer.

Second object of the present invention is to provide Computer-aided Diagnosis of Breast Cancer kit.

Inventor is by separating and studying patient with breast cancer and compareed in peripheral blood DNA with the healthy women of its age-matched SNP, find the high specific and the SNP of sensitiveness of one group and breast cancer height correlation, and develop can be just In the Computer-aided Diagnosis of Breast Cancer kit of clinical practice, supported for the examination and diagnosis of breast cancer provide data.

The purpose of the present invention is realized by following technical proposal：

One group of biomarker of auxiliary diagnosis breast cancer, the biomarker be SNP site rs114337127, rs13306048、rs139477122、rs140787599、rs16859487、rs199972750、rs200315000、 The combination of rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497.

Invention further provides the specificity amplification primer of the biomarker SNP site, these primer sequences For：

The amplimer sequence of rs114337127 is SEQ ID No：1 and SEQ ID No：2；

The amplimer sequence of rs13306048 is SEQ ID No：3 and SEQ ID No：4；

The amplimer sequence of rs139477122 is SEQ ID No：5 and SEQ ID No：6；

The amplimer sequence of rs140787599 is SEQ ID No：7 and SEQ ID No：8；

The amplimer sequence of rs16859487 is SEQ ID No：9 and SEQ ID No：10；

The amplimer sequence of rs199972750 is SEQ ID No：11 and SEQ ID No：12；

The amplimer sequence of rs200315000 is SEQ ID No：13 and SEQ ID No：14；

The amplimer sequence of rs35115195 is SEQ ID No：15 and SEQ ID No：16；

The amplimer sequence of rs368392829 is SEQ ID No：17 and SEQ ID No：18；

The amplimer sequence of rs369896558 is SEQ ID No：19 and SEQ ID No：20；

The amplimer sequence of rs372909717 is SEQ ID No：21 and SEQ ID No：22；

The amplimer sequence of rs373617497 is SEQ ID No：23 and SEQ ID No：24.

Further, present invention also offers the biomarker in the detection of breast cancer auxiliary, diagnosis, treatment and prognosis Application in assessment.

Further, the invention provides a kind of kit of Computer-aided Diagnosis of Breast Cancer, it includes detecting described SNP The reagent of point gene type, SNP site rs114337127, rs13306048, rs139477122, rs140787599, Rs16859487, rs199972750, rs200315000, rs35115195, rs368392829, rs369896558 and The combination of rs372909717, rs373617497.

Preferably, described reagent includes the specific primer for expanding the SNP site, or including for expanding The specific primer and restriction enzyme of the SNP site.

Preferably, the specific primer for expanding the SNP site is the combination of above-mentioned specific primer, and the specificity is drawn Thing combines high specificity, and expanding effect is good.

Preferably, the kit also includes the conventional enzyme and reagent of PCR reactions, such as dNTPs, Taq enzyme, Mg²⁺, PCR it is anti- Answer buffer solution etc.；Standard items and/or reference substance can also be contained.

Beneficial effect of the present invention：

Present invention research biomarker illustrates SNP for breast cancer progression in the application prospect of Computer-aided Diagnosis of Breast Cancer Influence, disclose its diagnostic value.Therefore, the present invention passes through the development of SNP gene type diagnostics reagent and diagnostic kit and answers With, may be such that the diagnosis of breast cancer is more convenient and easy, it is that clinician quick and precisely grasps conditions of patients, it is clinical treatment effect Fruit is evaluated and lays the foundation, and to find that the new small molecule drug targets with potential therapeutic value provide help.

Specific embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment In the conventional meanses that are well known to those skilled in the art of technological means used.

Technical scheme is specifically included：Gather standard compliant blood sample, the complete demography of systematic collection Data and clinical data；Genotype detection：Selection breast cancer case is compareed with the healthy women of breast cancer case age-matched, It is sequenced using extron, finds out the SNP related to breast cancer；To the positive association SNP for filtering out, further using Genotyping Detected, verified its repeatability for being applied to clinical diagnosis；The development of Computer-aided Diagnosis of Breast Cancer kit：According to breast cancer The genotype distribution frequency SNP that there were significant differences exploitation SNP auxiliary diagnostic boxes in case and healthy women control.

Each numerical value is expressed as follows in data analysis：

1、ljb23_sift：SIFT score values (version 2.3), represent influence of the variation to protein sequence, comprising three Individual value, one is SIFT initial values, and two is the value (1-SIFT) after conversion, and three is T or D.When the variation influences multiple simultaneously During protein sequence, there is a SIFT value to every protein sequence, take minimum value.SIFT score values are smaller more " harmful ", show the SNP The possibility for causing protein structure or function to change is big；D:Deleterious(sift<=0.05)；T:tolerated(sift> 0.05))；

2、ljb23_pp2hvar：Predict the variation to protein sequence based on HumanVar databases using PolyPhen2 Influence, for single gene inheritance disease.The row include two values, and first is PolyPhen2 score values, and numerical value is more big more " harmful ", Show that the SNP causes the possibility that protein structure or function change big；Second is D or P or B (D:Probably damaging (>=0.909), P:possibly damaging(0.447<=pp2_hvar<=0.909)；B:benign(pp2_hvar<= 0.446))；

3、ljb23_pp2hdiv：Predict the variation to protein sequence based on HumanDiv databases using PolyPhen2 Influence, for complex disease.The row include two values, and first is the score values of PolyPhen 2, and numerical value is more big more " harmful ", shows The SNP causes the possibility that protein structure or function change big；Second is D or P or B (D:Probably damaging(>= 0.957),P:possibly damaging(0.453<=pp2_hdiv<=0.956)；B:benign(pp2_hdiv<= 0.452))；

4、ljb23_mt：Mutation Taster score values (version 2.3), represent shadow of the variation to protein sequence Ring, comprising three values, one is Mutation Taster initial values, and two is the value after conversion, and three is A, D, N or P.Second It is individual to be worth more big more " harmful ", show that the SNP causes the possibility that protein structure or function change big, wherein " A " (" disease causing automatic")；"D"("disease causing")；"N"("polymorphism")；"P"(" polymorphism automatic")。

The experimental technique specifically studied mainly includes following components：

1. the selection of sample is studied

(1) breast cancer case 25 clarified a diagnosis through pathology and the healthy women 10 with breast cancer case age-matched Example wherein has 3 patients to have cancer family history as control in breast cancer case；

(2) take a blood sample before do not received radiotherapy or chemotherapy, without the past tumour medical history；

(3) healthy women with case age-matched is compareed

2. phenol-chloroform method extracts peripheral blood genomic DNA, operates according to a conventional method.Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D：2800D) in 1.6-2.0.

3. full Exon chip detection

(1) subject's complete genome DNA sample is taken；

(2) it is scanned on full Exon chip (Beijing Nuo Hezhi sources Science and Technology Co., Ltd., similarly hereinafter)；

(3) detect and difference difference of relatively more each genotype in breast cancer case is compareed with healthy women.

4. the Genotyping of single SNP

(1) subject's DNA sample is taken；

(2) specificity amplification primer of single SNP is designed；

(3) performing PCR reaction is entered, recovery product is sequenced；

(4) distributional difference of different genotype during breast cancer case is compareed with healthy women is compared.

5. diagnostic reagent box preparation method

Full Exon chip is scanned gene in being compareed with healthy women with determination breast cancer case after single SNP detections The type distribution frequency SNP that there were significant differences, as the index of breast cancer diagnosis.The SNP relevant with pathogenesis of breast carcinoma for filtering out Auxiliary diagnostic box, it include detection rs114337127, rs13306048, rs139477122, rs140787599, rs16859487、rs199972750、rs200315000、rs35115195、rs368392829、rs369896558、 The reagent of the SNP site genotype of rs372909717 and rs373617497, diagnostic kit can also include the spy of these SNP Specific amplification primers, and the reagent such as Taq enzyme, dNTPs.

6. clinical practice

Using the present inventor prepare Computer-aided Diagnosis of Breast Cancer kit detection treat examination patient with breast cancer and with reality Clinical detection compares that the validity of Computer-aided Diagnosis of Breast Cancer kit is determined.Specifically include measure subject's blood specimen The specificity amplification primer of above-mentioned SNP and other detection reagents in DNA, are disease shape that clinician quick and precisely grasps patient State and coincident with severity degree of condition, take the control prece of more personalized to provide support in time.

The collection of the sample of embodiment 1 and the arrangement of sample data

Inventor have collected substantial amounts of new hair-cream gland in January, 2010 in December, 2015 in Shenzhen City Second People's Hospital Cancer patient's blood specimen, by the arrangement to sample data, inventor therefrom have selected 25 samples for meeting following standard, while 10 ages of selection compare in 25-55 Sui healthy women and carry out full Exon chip detection, and sample selection criteria is as follows：

1st, the breast cancer case clarified a diagnosis through pathology, wherein there is 3 patients to there is cancer family history and mark respectively It is X1, X2, X3；

2nd, take a blood sample before do not received radiotherapy or chemotherapy, without the past tumour medical history；

3 compare with the healthy women of case age-matched

And situations such as demographic data and the clinical data of system acquisition these samples.

The extraction and purifying of the peripheral blood DNA of embodiment 2

In above-mentioned qualified 25 patient with breast cancers and 10 healthy women controls, two groups of age equilibriums are comparable.

Concretely comprise the following steps：

1st, add hemolyzing reagent to the peripheral blood that is stored in 2mL cryopreservation tubes (i.e. lysate, 40 deal collocation methods are such as Under：After sucrose 219.72g, magnesium chloride 2.02g and triton x-100 (amresco0694) 20mL mixing, TrisHcl solution is used 2000mL is settled to, similarly hereinafter), it is transferred to completely after reverse mixing.

2nd, red blood cell is removed：5mL centrifuge tubes are mended to 4mL with hemolyzing reagent, are overturned and is mixed, 4000rpm is centrifuged 10 minutes, Abandon supernatant.To 4mL hemolyzing reagents are added in precipitation, overturn mix cleaning once again, 4000rpm is centrifuged 10 minutes, abandons supernatant.

3rd, DNA is extracted：(contain 122.5mL0.2M sodium chloride, 14.4mL in per 300mL to 1mL extracts are added in precipitation 0.5M ethylenediamine tetra-acetic acids, 15mL10% lauryl sodium sulfate, 148.1mL distilled waters, similarly hereinafter) and 8 μ L Proteinase Ks, vibration Fully vibration is mixed on device, and 37 DEG C of water-baths are overnight.

4th, isolating protein is removed：Plus 1mL saturated phenols are fully mixed (hand jog 15 minutes), 4000rpm is centrifuged 10 minutes, takes It is transferred to clearly in new 5mL centrifuge tubes.Isometric chloroform and isoamyl alcohol mixed liquor (chloroform are added in supernatant：Isoamyl alcohol=24: 1, v/v, similarly hereinafter), after fully mixing (hand 15 minutes), 4000rpm is centrifuged 10 minutes, take supernatant (be divided into two 1.5mL from Heart pipe).

5th, DNA precipitations：The μ L of sodium acetate 60 of 3M are added in supernatant, the anhydrous second of the ice isometric with supernatant is added Alcohol, upper and lower jog, it is seen that white flock precipitate thing, then 10min is centrifuged with 12000rpm.

6th, DNA washings：Ice absolute ethyl alcohol 1mL, 12000rpm centrifugation 10min is added in precipitation, vacuum is taken out after abandoning supernatant Do or be placed in and be evaporated in cleaning dry environment.

7th, concentration is measured：Usually lead to 20-50ng/ μ LDNA, purity (ultraviolet 2600D：2800D) in 1.8-2.0.

The full extron group detection of embodiment 3SNP

Two groups of crowds in embodiment 2 are obtained into correlated results through the detection of full Exon chip.

1st, library construction

Beijing Nuo Hezhi sources Science and Technology Co., Ltd. uses the liquid-phase chip capture systems of Agilent, to the complete outer of people Aobvious subregion DNA carries out efficiently concentrating, and high flux, high depth sequencing are then carried out on IlluminaHiseq platforms.Build storehouse and Capture experiment uses Agilent SureSelectHumanAll ExonV5 kits, the reagent that strict operation instructions are recommended And consumptive material, and operated with reference to the newest experiment flow by optimization.

Experiment basic procedure：Genomic DNA is broken into the piece that length is 180-280bp at random through the broken instrument of Covaris Section, connects top connection and prepares DNA library respectively after repairing and add A tails through end at fragment two ends.Library with special index With up to 543 after pooling, 872 probes of biotin labeling carry out solution hybridization, and reusing the magnetic bead with streptomysin will 20,965 the 334 of gene, 378 exon trappings get off, qualified to enter through the laggard style of writing storehouse quality inspection of PCR linear amplifications Machine sequencing on row.

2nd, storehouse inspection

After the completion of library construction, first carried out using Qubit2.0 it is tentatively quantitative, dilution library to 1ng/ μ L, then use Agilent 2100 detects to the insert size in library, insert size meet it is expected after, use Q-PCR methods pair The valid density in library carries out accurate quantitative analysis (library valid density>2nM), ensureing Library Quality.

3rd, upper machine sequencing

Storehouse inspection is qualified, and valid density and data output demand according to library carry out the sequencing of Illumina Hiseq platforms.

4th, data analysis and treatment

It is final to determine " breast cancer by data screening, deep processing and bioinformatics sequence alignment and genetic analysis It is preferred that case " is organized with genotype distribution frequency 53 SNP sites that there were significant differences found in " healthy women control " group Sensitivity level site.Protein function prediction, SNP mutation are carried out using programs such as SIFT, PolyPhen-2 and Mutation Taster Site is as shown in table 1 to albumen influence value result：

1 12 gene SNP mutational sites of table are to albumen influence value

Sequence number

Gene is referred to as

dbSNP_RS

ljb23_sift

ljb23_pp2hvar

ljb23_pp2hdiv

ljb23_mt

1

NGFR

rs114337127

0.41,0.59,T

0.0,B

1.000,1.000,D

2

TBXA2R

rs13306048

0,1.00,D

.

1,0.0,N

3

KITLG

rs139477122

0.06,0.94,T

0.006,B

0.003,B

0.961,0.961,D

4

ADRM1

rs140787599

0.03,0.97,D

0.166,B

0.763,P

1,1.0,D

5

CYBRD1

rs16859487

0.07,0.93,T

0.998,D

1.0,D

1,1.0,D

6

ADAMTS10

rs199972750

0.13,0.87,T

0.3,B

0.914,P

1.000,1.000,D

7

KIAA1407

rs200315000

0.25,0.75,T

0.0,B

1,1.0,D

8

NRK

rs35115195

0,1.00,D

0.998,D

1.0,D

1,1.0,D

9

ARHGAP18

rs368392829

.

0.145,B

0.482,P

1,1.0,D

10

TBKBP1

rs369896558

0.19,0.81,T

0.049,B

0.147,B

0.670,0.670,D

11

CAPN15

rs372909717

0,1.00,D

0.998,D

1.0,D

1,1.0,D

12

ZNF598

rs373617497

0.04,0.96,D

0.99,D

1.0,D

1.000,1.000,D

These sites can confirm that to be breast cancer candidate markers by bioinformatic analysis.

Embodiment 4 further analyzes the onset risk of SNP and breast cancer using MELD method

The present inventor is by 2 groups of samples (" breast cancer case group " and " healthy women control group ") genotype distribution frequency Comparing, select positive association SNP, single SNP regression coefficients are further tried to achieve as weight with full extron scanned samples Dangerous score value, draws ROC to evaluate the sensitivity and specificity of diagnosis, and then diagnose judgements of these SNP to pathogenesis of breast carcinoma Ability.Conjoint Analysis discovery to all SNP marks, 13 SNP mutations of gene in table 1：rs114337127、 rs13306048、rs139477122、rs140787599、rs16859487、rs199972750、rs200315000、 Rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497, its sensitivity and specificity All reach more than 60%.

Therefore, inventors demonstrated that the site mark can well by healthy women control and patient with breast cancer area Point.

The Genotyping of the single SNP of embodiment 5

1st, 5 patient with breast cancers and 5 healthy women comparison DNA samples are taken with embodiment 2；

2nd, PCR amplifications

The online primer-design software and primer premier 5.0, https provided using NCBI websites:// www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgiLINK_LOC=BlastHo meAd are to 12 The specificity amplification primer that individual gene designs single SNP is as shown in table 2.

The primer sequence of table 2

PCR reaction systems are as shown in table 3.PCR amplification programs are：95 DEG C of predegeneration 10min, 94 DEG C of denaturation 15s, 58 DEG C- 68 DEG C of (the Tm values according to different primers are different) annealing 15s, 72 DEG C of extension 30s, carry out 30 circulations, last 72 DEG C of extensions 30min, in 4 DEG C of preservations, overnight needs to place -20 DEG C of freezings.

The reaction system of table 3

Component	Addition
		2×mix	25μL
Sense primer (10uM)	3.0μL
		Anti-sense primer (10uM)	3.0μL
Template	5μL
		Add sterile purified water	To 50 μ L

3rd, it is sequenced

After PCR amplifications terminate, 5 μ L amplified productions are taken, 1% agarose gel electrophoresis, electrophoresis 30min dyes 20min, so Gel piece is placed in gel imaging instrument is afterwards observed, according to the clip size situation for comparing Marker, tentatively judge amplified fragments It is whether correct.And then satisfactory amplified production is purified：Using Mag- BindOligonucleotidePurificationKit kits, and operated by kit requirement.Loading is sequenced：Using ABI companies BigDye3.1SequencingKit kits, and operated by kit requirement；Surveyed with the type of ABI companies 3730 Sequence instrument is sequenced.

4th, interpretation of result

By Chromas sequence analysis softwares, sequencing result is compared with standard sequence, find SNP site, passed through The type of base at analysis SNP site, it is possible to obtain the genotype of SNP site.Result shows that above-mentioned 12 SNP sites are true Real storage variation.

So as to further confirm that 12 SNP sites can be used for detection, treatment, diagnosis, prognosis evaluation of breast cancer etc. Auxiliary diagnosis.

Embodiment 6 is used for the making of Computer-aided Diagnosis of Breast Cancer SNP kits

Based on the primer sets that embodiment 5 is obtained, the kit for breast cancer of the present invention, the kit are assembled Including the specific primer for expanding 12 SNP sites, the kit can also have normal needed for corresponding round pcr With reagent, such as：DNTPs, MgCl₂, distilled water, Taq enzyme etc., these common agents be all it is well known to those skilled in the art, separately Can also there are standard items and control (such as determining the standard items and blank of genotype) outward.The value of this kit is only Need peripheral blood without other tissue samples, detect SNP with special primer pair by most simplifying, then composed by SNP auxiliary Judgement breast cancer is helped, is not only stablized, it is easy to detect, and accurately, the Sensitivity and Specificity of medical diagnosis on disease is greatly improved, therefore will The input practice of this kit, can help instruct diagnosis and more effective individualized treatment.

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, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Shenzhen City Second People's Hospital

<120>The biomarker of one group of auxiliary diagnosis breast cancer and its application

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Claims

1. one group of biomarker of auxiliary diagnosis breast cancer, it is characterised in that the biomarker is SNP site rs114337127、rs13306048、rs139477122、rs140787599、rs16859487、rs199972750、 The group of rs200315000, rs35115195, rs368392829, rs369896558, rs372909717 and rs373617497 Close.

2. the specificity amplification primer of biomarker SNP site described in claim 1, it is characterised in that these primer sequences For：

The amplimer sequence of rs114337127 is SEQ ID No：1 and SEQ ID No：2；

The amplimer sequence of rs13306048 is SEQ ID No：3 and SEQ ID No：4；

The amplimer sequence of rs139477122 is SEQ ID No：5 and SEQ ID No：6；

The amplimer sequence of rs140787599 is SEQ ID No：7 and SEQ ID No：8；

The amplimer sequence of rs16859487 is SEQ ID No：9 and SEQ ID No：10；

The amplimer sequence of rs199972750 is SEQ ID No：11 and SEQ ID No：12；

The amplimer sequence of rs200315000 is SEQ ID No：13 and SEQ ID No：14；

The amplimer sequence of rs35115195 is SEQ ID No：15 and SEQ ID No：16；

The amplimer sequence of rs368392829 is SEQ ID No：17 and SEQ ID No：18；

The amplimer sequence of rs369896558 is SEQ ID No：19 and SEQ ID No：20；

The amplimer sequence of rs372909717 is SEQ ID No：21 and SEQ ID No：22；

The amplimer sequence of rs373617497 is SEQ ID No：23 and SEQ ID No：24.

3. biomarker described in claim 1 and specificity amplification primer described in claim 2 in the detection of breast cancer auxiliary, examine Application in disconnected, treatment and prognosis evaluation.

4. a kind of kit of Computer-aided Diagnosis of Breast Cancer, it is characterised in that it includes SNP site base described in test right requirement 1 Because of the reagent of type, the reagent can detect rs114337127 in peripheral blood DNA, rs13306048, rs139477122, rs140787599、rs16859487、rs199972750、rs200315000、rs35115195、rs368392829、 The combination of rs369896558, rs372909717 and rs373617497.

5. kit as claimed in claim 4, it is characterised in that the reagent includes the spy for expanding the SNP site Specific primer, or including the specific primer and restriction enzyme for expanding the SNP site.

6. kit as claimed in claim 5, it is characterised in that the specific primer sequence has SEQID NO：1-24 Shown nucleotide sequence.

7. the kit as described in claim 5 or 6, it is characterised in that the kit also includes dNTPs, Taq enzyme, Mg²⁺With PCR reaction buffers.