CN109355392A - The diagnostic kit of breast cancer high risk gene and its application - Google Patents

The diagnostic kit of breast cancer high risk gene and its application Download PDF

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Publication number
CN109355392A
CN109355392A CN201811494525.XA CN201811494525A CN109355392A CN 109355392 A CN109355392 A CN 109355392A CN 201811494525 A CN201811494525 A CN 201811494525A CN 109355392 A CN109355392 A CN 109355392A
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seq
primer
breast cancer
kit
dna
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黄薇
牛振民
施锦绣
张晨辉
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Shanghai Industrial Institute For Research And Technology
Chinese National Human Genome Center at Shanghai
Shanghai Human Genome Research Center
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Shanghai Industrial Institute For Research And Technology
Shanghai Human Genome Research Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development

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  • Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Hospice & Palliative Care (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to the diagnostic kit of breast cancer high risk gene and its applications.The relevant representative multiple genes of breast cancer have been selected, have carried out the design of primers suitable for amplification based on these selected genes.The primer is by reasonable design, preferably obtains, and for all exons and exon introne join domain sequence of the interested gene of the present invention, also, it is good to be used for specificity when PCR amplification, and amplification efficiency height.Method of the invention can carry out breast cancer polygenes detection.The meaning of the polygenes detection is far not limited to the clinical recommendation carried out based on individual and its family, can more push the optimization of the clinical recommendation of screening for cancer or cancer prevention.

Description

The diagnostic kit of breast cancer high risk gene and its application
Technical field
The invention belongs to medical diagnosis on disease research fields, more particularly it relates to which breast cancer high risk genetic test tries Agent box and its application.
Background technique
Breast cancer is one of most common malignant tumour of Womankind Worldwide, accounts for the 23% of female cancer morbidity sum, accounts for women The 14% of number of cancer deaths.Metastasis in Breast Cancer recurrence is the important cause of death of patient, and median survival time is less than 2 after transfer Year.In China, the disease incidence of breast cancer is showed an increasing trend year by year in recent years.Since the nineties, Chinese pathogenesis of breast carcinoma Rate growth rate is the whole world more than twice, and urban area is especially pronounced.By taking Beijing and Shanghai as an example, breast cancer continuous 20 years Positioned at female malignant first, present annual morbidity has reached 40/,100,000 or more, and annual still with the speed of 2-3% Rise, has the tendency that gradually close to European and American developed countries' level.In addition, compared with European and American developed countries women, Chinese Breast Cancer Patient premenopausal patients account for more than half (American-European less than 30%), and age of onset peak is 5 to 10 years early compared with west women.By In the core force that the women of this age bracket is family and society, breast cancer has further been highlighted to Chinese society and family's band The significant damage come.Meanwhile Shanghai is one of the most city of Chinese population and the highest city of breast cancer incidence, sea City's pathogenesis of breast carcinoma trend represents the disease trend in following 20 years Urbanization Process In Chinas.
The pathogenesis of breast cancer does not understand completely so far.Family history of breast cancer, benign breast disease history, the later moon Through age of menarche, higher stress, higher body-mass index (Body Mass Index, BMI) and Family history of cancer Etc. being considered as the major incentive for suffering from breast cancer.The cause of disease of breast cancer for hereditary angle there has also been more breakthrough, A series of breast cancer susceptibility bases such as BRCA1, BRCA2, TP53, PTEN, STK11/LKB1, CDH1, CHEK2, ATM, MLH1, MSH2 The mutation of cause is also proved to related to breast cancer.
But there is still a need for the diagnostic reagents or kit that further optimize breast cancer for this field, with more not convenient and fast Technology realizes more accurate detection.
Summary of the invention
The purpose of the present invention is to provide the applications of breast cancer high risk gene detecting kit and the kit.
In the first aspect of the present invention, provide a kind of for detecting breast cancer risk or carrying out prognosis to breast cancer Reagent, the reagent are primer pairs, are selected from the group:
SEQ ID NO:1 and SEQ ID NO:2；
SEQ ID NO:3 and SEQ ID NO:4；
SEQ ID NO:5 and SEQ ID NO:6；
SEQ ID NO:7 and SEQ ID NO:8；
SEQ ID NO:9 and SEQ ID NO:10；
SEQ ID NO:11 and SEQ ID NO:12；
SEQ ID NO:13 and SEQ ID NO:14；
SEQ ID NO:15 and SEQ ID NO:16；
SEQ ID NO:17 and SEQ ID NO:18；
SEQ ID NO:19 and SEQ ID NO:20；
SEQ ID NO:21 and SEQ ID NO:22；
SEQ ID NO:23 and SEQ ID NO:24；
SEQ ID NO:25 and SEQ ID NO:26；
SEQ ID NO:27 and SEQ ID NO:28；
SEQ ID NO:29 and SEQ ID NO:30；
SEQ ID NO:31 and SEQ ID NO:32；
SEQ ID NO:33 and SEQ ID NO:34；
SEQ ID NO:35 and SEQ ID NO:36；
SEQ ID NO:37 and SEQ ID NO:38；
SEQ ID NO:39 and SEQ ID NO:40；
SEQ ID NO:41 and SEQ ID NO:42；
SEQ ID NO:43 and SEQ ID NO:44；
SEQ ID NO:45 and SEQ ID NO:46；
SEQ ID NO:47 and SEQ ID NO:48；
SEQ ID NO:49 and SEQ ID NO:50；
SEQ ID NO:51 and SEQ ID NO:52；
SEQ ID NO:53 and SEQ ID NO:54；
SEQ ID NO:55 and SEQ ID NO:56；
SEQ ID NO:57 and SEQ ID NO:58；
SEQ ID NO:59 and SEQ ID NO:60；
SEQ ID NO:61 and SEQ ID NO:62；
SEQ ID NO:63 and SEQ ID NO:64；
SEQ ID NO:65 and SEQ ID NO:66；
SEQ ID NO:67 and SEQ ID NO:68；
SEQ ID NO:69 and SEQ ID NO:70；
SEQ ID NO:71 and SEQ ID NO:72；
SEQ ID NO:73 and SEQ ID NO:74；
SEQ ID NO:75 and SEQ ID NO:76；
SEQ ID NO:77 and SEQ ID NO:78；
SEQ ID NO:79 and SEQ ID NO:80；
SEQ ID NO:81 and SEQ ID NO:82；
SEQ ID NO:83 and SEQ ID NO:84；
SEQ ID NO:85 and SEQ ID NO:86；
SEQ ID NO:87 and SEQ ID NO:88.
In a preferred embodiment, the gene that the primer is expanded be include gene selected from the group below: TP53, PTEN, STK11, CDH1 and PALB2.
In another aspect of this invention, the purposes of the reagent is provided, hybrid detection reagent is used to prepare；Or for making The standby kit containing the hybrid detection reagent；Or it is used to prepare detection breast cancer risk or prognosis is carried out to breast cancer Kit.
In another aspect of this invention, it provides a kind of for detecting breast cancer risk or carrying out prognosis to breast cancer Kit, containing being selected from one or more primer pairs above-mentioned in the kit.
In a preferred embodiment, the kit kind contains 10~44 kinds of primer pairs；More preferably for 20~44 kinds or 30~ 44 kinds of primer pairs.
In another aspect of this invention, it provides a kind of for detecting breast cancer risk or carrying out prognosis to breast cancer Kit, wherein containing hybrid detection reagent, the hybrid detection reagent is by 10~44 kinds of primer pairs in table 2；More preferably it is 20~44 kinds or 30~44 kinds of primer pairs mix.
In another preferred example, also contain in the kit: PCR amplification reagent.
In another preferred example, also contain in the kit: nucleic acid extracting reagent.
In another preferred example, also contain in the kit: Nucleotide Sequence Analysis Software.
In another preferred example, also contain in the kit: operation instructions.
In another aspect of this invention, it provides in a kind of determining determined nucleic acid sample with the presence or absence of breast cancer related gene Method, the method include: (1) using PCR amplification is carried out in primer pair determined nucleic acid sample above-mentioned, obtain amplification and produce Object；(2) gene order of (1) amplified production is analyzed.
In a preferred embodiment, in step (2), the analysis of gene order is carried out using sequenator.
In another preferred example, the method is in-vitro method.
In another preferred example, the method be not method for the purpose of obtaining medical diagnosis on disease result.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.
Specific embodiment
The present inventor passes through in-depth study, has selected the relevant representative multiple genes of breast cancer, is based on institute These genes of selection carry out the design of primers suitable for amplification.The primer is by reasonably designing, preferably obtaining, for this All exons and exon introne join domain sequence of interested gene are invented, also, are used for special when PCR amplification Property it is good, and amplification efficiency is high.Method of the invention can carry out breast cancer polygenes detection.The meaning of the polygenes detection is remote It is not limited to the clinical recommendation carried out based on individual and its family, can more push the clinical recommendation of screening for cancer and (or) cancer prevention Optimization.
The relevant gene of breast cancer diagnosis and primer
In this field, it is believed that gene groups gene that is various, such as being recited in table 1 relevant to breast cancer, in addition also There is more unlisted arrive but by the gene of those skilled in the art.
The present inventor has selected representative multiple breast cancer related genes, is carried out based on these selected genes Design of primers suitable for amplification.The gene be include gene selected from the group below: TP53, PTEN, STK11, CDH1 and PALB2。
Table 1
The present inventor has found under study for action, when detecting each gene mutation of the invention, carries out PCR using general primer Specificity is poor when amplification, and amplification efficiency is not high.Therefore, it is necessary to design and screen the good primer of specificity.By a large amount of real It tests and compares, the present inventor has found comparatively ideal 44 pairs of primers.Amplified production packet verified, that the primer obtains Representative sequence fragment is contained, and specificity is very good, PCR amplification success rate is close to 100%, especially suitable for complexity The PCR amplification of system.The nucleotide sequence of each primer is as shown in table 2 in embodiment.
Obtain the method and application of amplified production
Each gene of the invention can be detected using multiple technologies.It is with special as preferred embodiment of the invention Primer carry out PCR amplification, then to amplified production carry out sequencing, thus judge whether there is the gene and/or its Amount.The detection technique can both be directed to cDNA, can also be directed to genomic DNA.
Current inventor provides the sides that each gene amplification product of the invention is obtained in a kind of slave nucleic acid samples of optimization Method, the method include: to carry out PCR amplification using the primer pair selected from table 2 using nucleic acid samples as template, obtain amplification and produce Object.
Using the primer, ideal amplification can be obtained using conventional PCR amplification method.It is a kind of excellent In the PCR amplification method of choosing, steps are as follows for PCR thermal cycle: (a) carrying out 15 ± 2 seconds for 98 ± 1 DEG C；(b) 4 ± 1 are carried out for 60 ± 2 DEG C Minute；It repeats step (a)-(b) 18 ± 2 times.The side of this field routine can be used in template denaturation step before carrying out PCR thermal cycle Method.Preferable method are as follows: 95 ± 1 DEG C carry out 4 ± 1 minutes；More preferably 95 ± 0.5 DEG C carry out 4 minutes.
In addition to using primer of the invention, the present invention is to each ingredients other in PCR amplification system and its final concentration without spy Other limitation.Those skilled in the art can establish PCR amplification according to the general ingredient and its concentration that use when routinely establishing PCR system System.The conventional method that this field can also be used in template (such as genomic DNA) for PCR amplification, which is extracted, to be obtained.
The method that each gene amplification product of the invention is obtained using the present invention, amplification efficiency and the non-convention of specificity Think, and the PCR amplification particularly suitable for complex system, such as using poba gene group DNA as the amplification of pcr template.
The present invention also provides whether there is each gene of the invention and its presence in a kind of determining determined nucleic acid sample The method of amount.The method includes: that each gene piece of the invention is expanded from determined nucleic acid sample using method above-mentioned Section obtains amplified production；And in analysis amplified production each genetic fragment of the invention there are situation and amounts.
By judge each gene of the invention there are situation and amounts, can further learn the illness of subject Risk, to achieve the purpose that early monitoring early prevention and treatment.
It, can illness people to breast cancer based on the Close relation of the present inventor's selected each gene and breast cancer Group carries out susceptibility analysis, risk analysis or disease prognosis analysis, can also carry out early stage to the risk of correlated crowd Assessment.
In the preferred embodiment, the method that the present inventor uses the sequencing of two generations, using multiplexed PCR amplification come rich Collect all exons and exon introne join domain sequence of gene of interest, carry out deep sequencing, detects germline mutation.
Kit
The present invention also provides for detecting breast cancer risk or carrying out the kit of prognosis, the reagent to breast cancer Box includes: mentioned-above specific primer pair；Or including hybrid detection reagent, the hybrid detection reagent is by described 10 ~44 kinds of primer pairs；It is more preferably that 20~44 kinds or 30~44 kinds of primer pairs mix.
Can also it contain in the kit: nucleic acid extraction reagent (such as nucleic acid extraction liquid)；And/or polymerase chain reaction examination Agent (such as dNTP, Taq enzyme).For example, can contain: (A) various PCR reaction reagents, such as, but not limited to: Taq enzyme, PCR buffering Liquid, dNTP, archaeal dna polymerase etc.；Or reagent needed for (B) various extraction DNA or RNA (preparing PCR reaction template), such as but It is not limited to: phenol, chloroform, isoamyl alcohol, NaCl etc.；Or (C) extracts the kit of DNA or RNA.
Can also it contain in the kit as a preferred method: Nucleotide Sequence Analysis Software.
In addition, also containing the operation instructions and/or standard operation journey of kit of the invention in the kit Sequence.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.
Embodiment 1, PCR amplification and mastocarcinoma gene detection
Step 1 genome is quantitative
Genome is quantitative extremely important, usesDsDNA HS Assay Kit or quantitative fluorescent PCR are to genome Carry out accurate quantification.Specifically quantitatively process is joined2.0dsDNA quantitative approach specification.
The amplification of step 2 target region
Respectively using Panel Mix A, Panel Mix B as primer, according to reaction system listed below and amplification condition into Row first round PCR amplification obtains 2 pipe amplified productions.
PCR is 25 μ l reaction systems, in which:
PCR program:
The purifying of step 3 PCR product
AMPure XP Beads purifying magnetic bead is utilized respectively to purify 2 pipe PCR products.The purifying can be effective rich Collect target area segment, lowers non-specific amplification concentration.
1. 0.5 times of volume AMPure XP Beads (30 μ L systems add 15 μ L) is added to PCR reaction solution/enzymatic reaction solution, It is blown and beaten up and down with pipettor, mixes well amplified production with AMPure XP Beads, be stored at room temperature 2 minutes.
2. magnetic bead is adsorbed with strong magnets or magnetic frame, until solution clarification.
3. carefully drawing supernatant with pipettor (retains supernatant) into new EP pipe/96 orifice plates, carefully avoid being drawn onto magnetic Pearl.Magnetic bead in the step can be abandoned.
4. be added into supernatant 0.6 times of original PCR volume AMPure XP Beads (such as PCR system is 30 μ L, then plus 18 μ L magnetic beads), it is blown and beaten up and down with pipettor, mixes well amplified production with AMPure XP Beads.It is stored at room temperature 2 minutes.
5. magnetic bead is adsorbed with strong magnets or magnetic frame, until solution clarification.Supernatant is carefully drawn with pipettor, is thrown Supernatant is abandoned, magnetic bead is retained.
6. 40 μ L BW10 are added, suspension magnetic bead is stored at room temperature 2min.Magnetic bead is adsorbed with strong magnets or magnetic frame, until Until solution is clarified.Supernatant is drawn with pipettor, abandons supernatant, retains magnetic bead.
7. 100 μ L, 70% ethyl alcohol is added, magnetic bead is adsorbed back and forth on different two sides repeatedly with magnetic frame with the magnetic that sufficiently suspends Pearl is just washed.
8. magnetic bead is adsorbed with magnet or magnetic frame, until solution clarification.Supernatant is carefully removed with pipettor, avoids inhaling To magnetic bead.
9. being placed at room temperature for, until ethyl alcohol volatilizees completely.Magnetic bead can be also put in 50 DEG C of baking ovens 5 minutes by this step, rapid steaming Dry ethyl alcohol.This step can never be omitted, and otherwise remaining ethyl alcohol can seriously affect yield and subsequent experimental.
Step 4 second takes turns PCR
The second wheel PCR amplification is carried out to 2 pipe recovery products respectively.This step main purpose be introduce be sequenced corresponding connector with barcode。
PCR is 20 μ l reaction systems, in which:
PCR program:
The recycling of step 5 PCR product
2 pipe PCR products are purified using AMPure XP Beads purifying magnetic bead.
In 1.PCR product plus 0.8 times of volume AMPure XP Beads (30 μ L systems add 24 μ L) pipettor is blown up and down It beats, mixes well recovery product with AMPure XP Beads.It is stored at room temperature 2 minutes.
2. magnetic bead is adsorbed with strong magnets or magnetic frame, until solution clarification.
3. carefully drawing supernatant with pipettor, supernatant is abandoned, retains magnetic bead.
4. 40 μ L BW07 bead suspensions are added, it is vortexed uniform.
5. magnetic bead is adsorbed with magnet or magnetic frame, until solution clarification.Supernatant is carefully removed with pipettor, avoids inhaling To magnetic bead.
6. 100 μ L, 70% ethyl alcohol is added, magnetic bead is adsorbed back and forth on different two sides repeatedly with magnetic frame with the magnetic that sufficiently suspends Pearl is just washed.
7. magnetic bead is adsorbed with magnet or magnetic frame, until solution clarification.Supernatant is carefully removed with pipettor, avoids inhaling To magnetic bead.
8. being placed at room temperature for, until ethyl alcohol volatilization is clean.This step magnetic bead also can be placed on 50 DEG C of baking ovens 5 minutes or so, quickly It is evaporated ethyl alcohol.
9. 20 μ L Elution Buffer, sufficiently suspension magnetic bead are added, 2min is stored at room temperature with eluted dna.Magnetic bead is used Magnet absorption, obtained supernatant DNA solution are drawn to new 1.5/0.5/0.2mL centrifuge tube/96 hole PCR pipes.Elution Buffer is 10mM Tris-HCl, and pH 8.0-8.5 can also be replaced with TE.
10. DNA concentration measurement is carried out to two pipe purified product of A, B respectively, according to measurement concentration etc. than mixing two pipes purifying Product, mix products can be directly used for machine sequencing or are placed in -20 DEG C of preservations.
Table 2
Using above-mentioned detection reagent of the invention and detection method, 20 patients of clinic are detected and for a long time with Track observation, as a result, it has been found that, detection method accuracy of the invention is ideal.
It should be understood that after reading the above teachings of the present invention, those skilled in the art can make the present invention Various changes or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
Sequence table
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<211> 23
<212> DNA
<213>primer (Primer)
<400> 42
acacgagcag agaatcataa ggc 23
<210> 43
<211> 22
<212> DNA
<213>primer (Primer)
<400> 43
tctgaaagcg gctgatactg ac 22
<210> 44
<211> 23
<212> DNA
<213>primer (Primer)
<400> 44
gatttctgca tttcccagca cat 23
<210> 45
<211> 24
<212> DNA
<213>primer (Primer)
<400> 45
gtacatccaa gatcagtggt gcta 24
<210> 46
<211> 27
<212> DNA
<213>primer (Primer)
<400> 46
ccaacattgg tcttttgtga aatggtc 27
<210> 47
<211> 23
<212> DNA
<213>primer (Primer)
<400> 47
tttttgtcca gccagcaaat gag 23
<210> 48
<211> 27
<212> DNA
<213>primer (Primer)
<400> 48
ttttgggaac atggttttga ccttttt 27
<210> 49
<211> 22
<212> DNA
<213>primer (Primer)
<400> 49
tgcacagtgc ctttcagaat gt 22
<210> 50
<211> 30
<212> DNA
<213>primer (Primer)
<400> 50
gccagatctt tatttttcct gacatactct 30
<210> 51
<211> 29
<212> DNA
<213>primer (Primer)
<400> 51
gaaggtttgt tcattactgc ttatgactt 29
<210> 52
<211> 30
<212> DNA
<213>primer (Primer)
<400> 52
acctcctaag acatgctatg atgaataaga 30
<210> 53
<211> 29
<212> DNA
<213>primer (Primer)
<400> 53
tcttcacaac aaccctgtaa aattagagg 29
<210> 54
<211> 22
<212> DNA
<213>primer (Primer)
<400> 54
cggagaaggg ctacctagag ac 22
<210> 55
<211> 22
<212> DNA
<213>primer (Primer)
<400> 55
catttgctga gcctctcacc tt 22
<210> 56
<211> 28
<212> DNA
<213>primer (Primer)
<400> 56
tgatcaacaa gtagaagtca tgacgttt 28
<210> 57
<211> 32
<212> DNA
<213>primer (Primer)
<400> 57
agttaaaaat caatcaatgc ttttcttacc ct 32
<210> 58
<211> 30
<212> DNA
<213>primer (Primer)
<400> 58
aatattaaaa ggttactcct cacatcaccc 30
<210> 59
<211> 23
<212> DNA
<213>primer (Primer)
<400> 59
accagctgac agagacaaag atg 23
<210> 60
<211> 30
<212> DNA
<213>primer (Primer)
<400> 60
gagccttcaa atgatgaaaa ttatccttgt 30
<210> 61
<211> 22
<212> DNA
<213>primer (Primer)
<400> 61
ggcaaatggc tgcaaagatc tc 22
<210> 62
<211> 29
<212> DNA
<213>primer (Primer)
<400> 62
catacttatg ctttgcataa aacagcact 29
<210> 63
<211> 30
<212> DNA
<213>primer (Primer)
<400> 63
ccaaatctgt ttttctgaat ctgtttacca 30
<210> 64
<211> 25
<212> DNA
<213>primer (Primer)
<400> 64
gggtaatgca ggcagacatt ataca 25
<210> 65
<211> 30
<212> DNA
<213>primer (Primer)
<400> 65
gaaatcatat caaagaaaca cgtcaaacca 30
<210> 66
<211> 29
<212> DNA
<213>primer (Primer)
<400> 66
cacccattga gtcattcact tttaaagaa 29
<210> 67
<211> 22
<212> DNA
<213>primer (Primer)
<400> 67
cagctcctgg catgtgtttc ta 22
<210> 68
<211> 22
<212> DNA
<213>primer (Primer)
<400> 68
cagaagttgc tggacgaact tg 22
<210> 69
<211> 27
<212> DNA
<213>primer (Primer)
<400> 69
agactgagtc tttcaaatga gcaagtt 27
<210> 70
<211> 27
<212> DNA
<213>primer (Primer)
<400> 70
ttctaccagg aaaatcacat cccaaaa 27
<210> 71
<211> 22
<212> DNA
<213>primer (Primer)
<400> 71
ccgtctttgt atgctggctt tg 22
<210> 72
<211> 28
<212> DNA
<213>primer (Primer)
<400> 72
tacctgatga agactttgga cctcttaa 28
<210> 73
<211> 23
<212> DNA
<213>primer (Primer)
<400> 73
tggtttttct gagcaggact tca 23
<210> 74
<211> 30
<212> DNA
<213>primer (Primer)
<400> 74
gattgtctgt tttgttgggt tttgttacta 30
<210> 75
<211> 23
<212> DNA
<213>primer (Primer)
<400> 75
acacttggcc ctgtcacttt tta 23
<210> 76
<211> 24
<212> DNA
<213>primer (Primer)
<400> 76
tagcctgtcg attgttaaca ggtc 24
<210> 77
<211> 24
<212> DNA
<213>primer (Primer)
<400> 77
cgtgctgata tttgtgtgag gtga 24
<210> 78
<211> 26
<212> DNA
<213>primer (Primer)
<400> 78
caaccaagtt caagaacctc tcagaa 26
<210> 79
<211> 22
<212> DNA
<213>primer (Primer)
<400> 79
gcgggagagc tgactttagt ta 22
<210> 80
<211> 23
<212> DNA
<213>primer (Primer)
<400> 80
tccaattgcc agaggaaagt agc 23
<210> 81
<211> 28
<212> DNA
<213>primer (Primer)
<400> 81
ttcttgacat ccaaatgact ctgaatga 28
<210> 82
<211> 30
<212> DNA
<213>primer (Primer)
<400> 82
ggaatgaaaa tcttcaggaa agtgagattc 30
<210> 83
<211> 23
<212> DNA
<213>primer (Primer)
<400> 83
gttgcttcca ggctaagact ctt 23
<210> 84
<211> 26
<212> DNA
<213>primer (Primer)
<400> 84
actgtctcta cagataacct ccttgt 26
<210> 85
<211> 23
<212> DNA
<213>primer (Primer)
<400> 85
gggcagttgg ccacttttac tta 23
<210> 86
<211> 28
<212> DNA
<213>primer (Primer)
<400> 86
caaccagaaa aaggtgttga tacattcc 28
<210> 87
<211> 22
<212> DNA
<213>primer (Primer)
<400> 87
ctgtagtcgc cctggtgaaa tt 22
<210> 88
<211> 23
<212> DNA
<213>primer (Primer)
<400> 88
tgtctttggc actgattcac tca 23

Claims (10)

1. a kind of reagent for detecting breast cancer risk or carrying out prognosis to breast cancer, which is characterized in that the examination Agent is primer pair, including primer pair selected from the group below:
SEQ ID NO:1 and SEQ ID NO:2；
SEQ ID NO:3 and SEQ ID NO:4；
SEQ ID NO:5 and SEQ ID NO:6；
SEQ ID NO:7 and SEQ ID NO:8；
SEQ ID NO:9 and SEQ ID NO:10；
SEQ ID NO:11 and SEQ ID NO:12；
SEQ ID NO:13 and SEQ ID NO:14；
SEQ ID NO:15 and SEQ ID NO:16；
SEQ ID NO:17 and SEQ ID NO:18；
SEQ ID NO:19 and SEQ ID NO:20；
SEQ ID NO:21 and SEQ ID NO:22；
SEQ ID NO:23 and SEQ ID NO:24；
SEQ ID NO:25 and SEQ ID NO:26；
SEQ ID NO:27 and SEQ ID NO:28；
SEQ ID NO:29 and SEQ ID NO:30；
SEQ ID NO:31 and SEQ ID NO:32；
SEQ ID NO:33 and SEQ ID NO:34；
SEQ ID NO:35 and SEQ ID NO:36；
SEQ ID NO:37 and SEQ ID NO:38；
SEQ ID NO:39 and SEQ ID NO:40；
SEQ ID NO:41 and SEQ ID NO:42；
SEQ ID NO:43 and SEQ ID NO:44；
SEQ ID NO:45 and SEQ ID NO:46；
SEQ ID NO:47 and SEQ ID NO:48；
SEQ ID NO:49 and SEQ ID NO:50；
SEQ ID NO:51 and SEQ ID NO:52；
SEQ ID NO:53 and SEQ ID NO:54；
SEQ ID NO:55 and SEQ ID NO:56；
SEQ ID NO:57 and SEQ ID NO:58；
SEQ ID NO:59 and SEQ ID NO:60；
SEQ ID NO:61 and SEQ ID NO:62；
SEQ ID NO:63 and SEQ ID NO:64；
SEQ ID NO:65 and SEQ ID NO:66；
SEQ ID NO:67 and SEQ ID NO:68；
SEQ ID NO:69 and SEQ ID NO:70；
SEQ ID NO:71 and SEQ ID NO:72；
SEQ ID NO:73 and SEQ ID NO:74；
SEQ ID NO:75 and SEQ ID NO:76；
SEQ ID NO:77 and SEQ ID NO:78；
SEQ ID NO:79 and SEQ ID NO:80；
SEQ ID NO:81 and SEQ ID NO:82；
SEQ ID NO:83 and SEQ ID NO:84；
SEQ ID NO:85 and SEQ ID NO:86；
SEQ ID NO:87 and SEQ ID NO:88.
2. reagent as described in claim 1, which is characterized in that the gene that the primer is expanded be include selected from the group below Gene: TP53, PTEN, STK11, CDH1 and PALB2.
3. the purposes of reagent described in claim 1 is used to prepare hybrid detection reagent；Or it is used to prepare and contains the hybrid detection The kit of reagent.
4. the purposes of reagent described in claim 1 is used to prepare detection breast cancer risk or carries out prognosis to breast cancer Kit.
5. a kind of kit for detecting breast cancer risk or carrying out prognosis to breast cancer, which is characterized in that described Primer pair in kit containing claim 1；Or
Contain hybrid detection reagent in the kit, the hybrid detection reagent is drawn by 10~44 kinds in claim 1 Object pair；It is more preferably that 20~44 kinds or 30~44 kinds of primer pairs mix.
6. kit as claimed in claim 5, which is characterized in that the kit kind contains 10~40 kinds of primer pairs；More preferably Ground is 20~44 kinds or 30~44 kinds of primer pairs.
7. kit as claimed in claim 5, which is characterized in that also contain in the kit:
PCR amplification reagent, and/or
Nucleic acid extracting reagent.
8. kit as claimed in claim 5, which is characterized in that also contain in the kit
Nucleotide Sequence Analysis Software；And/or
Operation instructions.
9. whether there is the method for breast cancer related gene in a kind of determining determined nucleic acid sample, which is characterized in that the side Method includes:
(1) using PCR amplification is carried out in the primer pair determined nucleic acid sample of claim 1, amplified production is obtained；
(2) gene order of (1) amplified production is analyzed.
10. method as claimed in claim 9, which is characterized in that in step (2), point of gene order is carried out using sequenator Analysis.
CN201811494525.XA 2018-12-07 2018-12-07 The diagnostic kit of breast cancer high risk gene and its application Pending CN109355392A (en)

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Application publication date: 20190219