CN107245523A - Molecular probe and detection kit for breast cancer fluorescence in situ hybridization detection - Google Patents
Molecular probe and detection kit for breast cancer fluorescence in situ hybridization detection Download PDFInfo
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Abstract
A kind of molecular probe for breast cancer fluorescence in situ hybridization detection and the detection kit for including the molecular probe, the molecular probe is respectively：Seq ID NO in ER probes, its sequence such as sequence table:Shown in 1 sequence；Seq ID NO in PR probes, its sequence such as sequence table:Shown in 2 sequences；Seq ID NO in the probes of HER 2, its sequence such as sequence table:Shown in 3 sequences；Seq ID NO in EPCR probes, its sequence such as sequence table:Shown in 4 sequences；Seq ID NO in the probes of Ki 67, its sequence such as sequence table:Shown in 5 sequences.This fluorescence in situ hybridization detection kit is used for breast cancer detection, it is easy to detect, quickly, credible result.
Description
Technical field
The invention belongs to medical science, and in particular to a kind of molecule for breast cancer fluorescence in situ hybridization detection is visited
Pin and detection kit.
Technical background
Breast cancer is the great public health problem of today's society.In the world, the incidence of disease of breast cancer is from 20 generation
Recording, it is in rising trend always to start the end of the seventies.China is not the country occurred frequently of breast cancer, but in recent years, China's breast cancer hair
The growth rate of sick rate is really higher by national 1-2 percentage points occurred frequently.According to National Cancer Center and prevention and control of diseases office of the Ministry of Public Health
The pathogenesis of breast carcinoma data in 2009 announced for 2012 are shown：It is pernicious that national tumour registration area breast cancer incidence occupies women
The 1st of tumour, female mammary gland cancer morbidity (rough and careless) whole nation adds up to 42.55/10 ten thousand, and city is 51.91/10 ten thousand, rural area
For 23.12/10 ten thousand.
Halsted proposes radical mastectomy first within 1894, develops by the technologies of more than 100 years, to the synthesis of today
Treatment, the methods for diagnosis and treatment of breast cancer is improved constantly.But its higher incidence of disease and fatal rate still seriously threaten greatly
The health of some women.With the transformation and renewal for the treatment of concept, the treatment of breast cancer enters the complex treatment stage, and is formed
The Therapeutic mode that local treatment and whole body therapeutic are laid equal stress on.Clinically, according to the physical condition by stages with patient of tumour, synthesis is examined
Consider and use operation, radiotherapy, chemotherapy, a variety for the treatment of means such as endocrine therapy, Biological target therapy.And in these treatment methods
In, become important medical reference index during breast cancer diagnosis to the detection and discriminating of tumor markers.Help to carry
Early diagnosis finds the morbidity of breast cancer, the prognosis of more effective accurately estimation patient with breast cancer, and different patients are carried out
Personalized treatment.
Research in recent years shows that estrogen secretion is excessive and long term is a big factor of pathogenesis of breast carcinoma, and confirms
Estrogen may reside in breast cancer cell.Therefore, clinically, to ERs (ER) and progesterone receptor (PR)
Etiologic diagnosis, the important indicator as Prognosis in Breast Cancer and endocrine therapy.Show according to correlative study, chemotherapy can be controlled preferably
Treat the patient with breast cancer of estrogen receptor negative.HER- 2 oncogene is located on chromosome 17q21, and its gene outcome is a kind of
Transmembrane tyrosine kinase acceptor, this receptor is coupled by way of dimer, activation signal Signal Transduction Pathways, promotes the generation of tumour
Development.Therefore it is that important one kind is examined that selection HER-2, which is label and accurately detection and interpretation are carried out to patient HER-2 states,
Treatment means.EPCR is a kind of tumour cell endothelial protein C receptor, and there are some researches show MK can promote breast by EPCR/PAR1 paths
Gland cancer MDA-MB-231 cells in vitro angiogenesis, therefore the signal of interest path that EPCR is produced as breast cancer, can be selected
EPCR carries out the diagnosis of breast cancer by the detection of expression to EPCR as target spot.Ki-67 antigens are division growths in nucleus
GAP-associated protein GAP, is expressed in the G1 phases of cell cycle, S phases and G2 phases, and is not expressed then in G0 phases and other cell cycles, its table
The cell quantity in mitosis period can be pointed out up to level, is an independent predictive factor of Prognosis in Breast Cancer.Have
Research shows, Luminal Type B breast cancer has the features such as HER-2 positive, Ki-67 height expression, itself and the Luminal A types made
Breast cancer, which is compared, has the relative resistance of Endocrine treatment, the features such as clinical prognosis is poor.Label is in mammary gland in view of the above
The characteristics of tumor pathogenesis, with reference to fluorescence in situ hybridization technique, set up and a kind of be directed to breast cancer including a variety of effectors
Diagnosis and treatment detection method, with important research and clinical meaning, can aid in doctor more quickly and accurately to related patient
The diagnosis and treatment of body.
The content of the invention
It is an object of the invention to overcome the accuracy deficiency and effector of the existing detection kit for breast cancer
The irrational defect of selection of target spot includes five kinds of effector target spots there is provided one kind, easy to detect in actual clinical, quickly,
The fluorescence in situ hybridization detection kit of credible result, is used for breast cancer detection by the detection kit, and fluorescence can be overcome former
Difficulty of the position hybridization technique in terms of breast cancer diagnosis.
The present invention is achieved through the following technical solutions above-mentioned purpose：
A kind of molecular probe for breast cancer fluorescence in situ hybridization detection, including the one or more in following probe：
Seq ID NO in ER probes, its sequence such as sequence table:Shown in 1 sequence；
Seq ID NO in PR probes, its sequence such as sequence table:Shown in 2 sequences；
Seq ID NO in HER-2 probes, its sequence such as sequence table:Shown in 3 sequences；
Seq ID NO in EPCR probes, its sequence such as sequence table:Shown in 4 sequences；
Seq ID NO in Ki-67 probes, its sequence such as sequence table:Shown in 5 sequences.
A kind of detection kit of the molecular probe comprising described in claim 1, its have hybridization solution (X2), denaturing liquid and
DAPI counterstains.
Further：
The hybridization solution contains five kinds of specific probes, is respectively：Seq ID NO in ER probes, its sequence such as sequence table:
Shown in 1：Seq ID NO in PR probes, its sequence such as sequence table:Shown in 2：HER-2 probes, its sequence is as in sequence table
Seq ID NO:Shown in 3：Seq ID NO in EPCR probes, its sequence such as sequence table:Shown in 4：Ki-67 probes, its sequence is such as
Seq ID NO in sequence table:Shown in 5.
The hybridization solution is divided into two pipes, and often pipe is containing three kinds of probes in five kinds of specific probes, and wherein EPCR is two
All contain in pipe, other probes only contain in a pipe.
The hybridization solution is included：10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/v) formamide, 0.1% (w/
V) sodium pyrophosphate, 0.2% (w/v) PVP, 5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM
Tris/HCl(pH7.5)；And respectively five kinds of specific probes of 30pM fluorescence labeling.
The main component of the DAPI counterstains is DAPI (4,6- diamidino -2- benzene indole hydrochloride) and anti-colour fading liquid.
DAPI (4,6- diamidino -2- benzene indole hydrochloride) concentration is 4mg/ml.
Detection kit also includes the packing box for separating and concentrating packaging reagent bottle or pipe.
The present invention is provided to the special molecular probe of breast cancer fluorescence in situ hybridization detection, and a kind of FISH
Detection kit, including five kinds of effector target spots, are used for breast cancer detection, the detection side in actual clinical by the detection kit
Just, quickly, credible result, can overcome difficulty of the existing fluorescence in situ hybridization technique in terms of breast cancer diagnosis.The detection
The sample that kit can be detected is relatively broad, including but not limited to breast cancer patients fresh food frozen cancerous issue, culture it is attached
Cell, fixed frozen cell, PMBC, paraffin section etc..
Brief description of the drawings
Fig. 1 breast cancer lesion histotomy fluorescence in situ hybridization detection results.
Embodiment
The present invention is further described in detail below with reference to example and accompanying drawing.And state：Below explanation be only pair
Claimed is technical scheme for example, not any limitation to these technical schemes.The protection of the present invention
Scope is defined by the content that appended claims are recorded.
In one embodiment, a kind of molecular probe for breast cancer fluorescence in situ hybridization detection, including following probe
In one or more：
ER probes, its sequence is as follows：
5’-ACACGGTGCGCGAGGCCGGCCCGCCGGCATTCTACAGGCCAAATTCAGATAATCGACGCCAGGGTG
GCAGAGAAAGATTGGCCAGTACCAATGACAAGGGAAGTATGGCTATGGAATCTGCCAAGGAGACTCGCTACTGTGCA
GT-3 ', corresponding to the Seq ID NO in sequence table:1 sequence；
PR probes, its sequence is as follows：
5’-ACTGCTGTGTCGCCCAGCCGCAGGTCCGTTCCCGGGGAGCCAGACCTCGGACACCTTGCCTGAAGT
TTCGGCCATACCTATCTCCCTGGACGGGCTACTCTTCCCTCGGCCCTGCCAGGGAC A-3 ', corresponding in sequence table
Seq ID NO:2 sequences；
HER-2 probes, its sequence is as follows：
5’-GAGGTGCAGGGCTACGTGCTCATCGCTCACAACCAAGTGAGGCAGGTCCCACTGCAGAGGCTGCGG
ATTGTGCGAGGCACCCAGCTCTTTGAGGACAACTATGCCCTGGCCGTGCTAGACAA TGGAGACC-3 ', corresponding to sequence
Seq ID NO in list:3 sequences；
EPCR probes, its sequence is as follows：
5’-CTCCAAAGACTTCATATGCTCCAGATCTCCTACTTCCGCGACCCCTATCACGTGTGGTACCAGGGC
AACGCGTCGCTGGGGGGACACCTAACGCACGTGCTGGAAGGCCCAGACACCAACAC CACGATCA-3 ', corresponding to sequence
Seq ID NO in list:4 sequences；
Ki-67 probes, its sequence is as follows：
5’-TAGTCCTAGGAAAACTCCAGTTGCCAGTGATCAACGCCGTAGGTCCTGCAAAACAGCCCCTGCTTC
CAGCAGCAAATCTCAGACAGAGGTTCCTAAGAGAGGAGGGAGAAAGAGTGGCAACCTGCCTTCAAAGAGAGTGTCTA
TC-3 ', corresponding to the Seq ID NO in sequence table:5 sequences.
In one embodiment, a kind of detection kit for including the molecular probe, it has hybridization solution (X2), denaturation
Liquid and DAPI counterstains.The hybridization solution contains five kinds of specific probes, is respectively：Seq in ER probes, its sequence such as sequence table
ID NO:Shown in 1：Seq ID NO in PR probes, its sequence such as sequence table:Shown in 2：HER-2 probes, such as sequence table of its sequence
In Seq ID NO:Shown in 3：Seq ID NO in EPCR probes, its sequence such as sequence table:Shown in 4：Ki-67 probes, its sequence
Seq ID NO in row such as sequence table:Shown in 5.
In a preferred embodiment, the hybridization solution is divided into two pipes, and often pipe contains three kinds in five kinds of specific probes
Probe, wherein EPCR all contain in two pipes, and other probes only contain in a pipe.
In a preferred embodiment, the hybridization solution is included：10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/
V) formamide, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) PVP, 5mM Na2EDTA, 0.1% (v/v)
TritonX-100,50mM Tris/HCl (pH7.5)；And respectively five kinds of specific probes of 30pM fluorescence labeling.
In a preferred embodiment, the main component of the DAPI counterstains is DAPI (4,6- diamidino -2- benzene indoles salt
Acid) and anti-colour fading liquid.
In a preferred embodiment, DAPI (4,6- diamidino -2- benzene indole hydrochloride) concentration is 4mg/ml.
Example 1：The preparation of specific probe
Breast cancer correlation ER, PR, HER-2, EPCR, Ki-67 gene information reported according to NCBI, downloads correlated series profit
With databases such as SMART, and the software such as DNAMAN carries out analysis and homologous sequence is compared, and obtains the conservative region of the gene, with
Each gene order is target sequence, and five groups of markers for breast cancer gene by fluorescence probes are designed using the primer-design softwares of Oligo 6.0.
Probe sequence is subjected to Blastn comparisons in ncbi database, to verify that probe and other sequences are heterologous, so as to avoid hybridization from walking
Rapid non-specific amplification.All probes can be synthesized by Synesis Company.
The sequence of five oligonucleotide probes described above is listed in the end of writing.
The preparation of fluorescence labeling probe：From ROX, FAM, CY5 fluoresceins, conceptual design is that five kinds of probes are divided into two groups：
First group is ER, PR, EPCR；Second group is HER-2, EPCR, Ki-67, and every group of three probe ROX, FAM, CY5 fluoresceins are adopted
The mark respectively of probe is carried out with nick-translation.Fluorescein is directly covalently tied with probe nucleotide with phosphopentose skeleton
Close.Comprise the following steps that：
1. PCR purified products are dissolved into final concentration of 1g/L using TE buffer solutions.
2. prepare containing 1 μ l concentration be 3mg/ml DNA enzymatic storing liquid and 10 × DNA enzymatic solution, volume is 1ml.
3. mixing after the completion of nick translation reaction system, sample-adding, 37 DEG C of standing 12min are prepared in ice bath.Specific reactant
System is as follows：
Table one：Reaction system prepared by fluorescence labeling probe
| DNA probe | 8μl |
| 10 × NT buffer solutions | 10μl |
| 10μg/mlBSA | 5μl |
| The solution of 10 μ g/ml DNA enzymatics I | 10μl |
| DNTP (contains each 1mM of dATP, dGTP, dCTP) | 5μl |
| FITC (final concentration of 60-70 μM) | 8μl |
| Sterilize tri-distilled water | 54μl |
| React cumulative volume | 100μl |
4. the e. coli dna polymerase that 2.5 μ l concentration are 10U/ μ l is added.
5. PCR amplification instrument is put, 15 DEG C of 12 hours of incubation, 70 DEG C are denatured 5min 4 DEG C 5 hours.
6. 4 μ l 0.2M EDTA (pH=8.0) terminating reaction is added, ethanol precipitation is finally suspended with cot-1DNA and precipitated
Powder.
7. TAE agarose gel electrophoresis detection probe mark result, makees purification process to the product after mark and determines it
OD values, the incorporation efficiency of fluorescein and the concentration of probe are calculated by formula.
8. preserve：Plus isometric glycerine, -20 DEG C of packing storages.
Example 2：The preparation of detection kit
Hybridization solution is constituted：10% (w/v) dextran sulfate, 10mM NaCl, 20% (v/v) formamide, 0.1% (w/v) is burnt
Sodium phosphate, 0.2% (w/v) PVP, 5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM
Tris/HCl(pH7.5)；Respectively five kinds of specific probes (prepared by embodiment 1) of 30pM fluorescence labeling.
The preparation of DAPI counterstains：2.2mgDAPI powder is taken, 550 μ l distilled water is added and mixes, DAPI stostes 4mg/ is made
ml.4 DEG C of preservations.Working solution：Take 2.5 μ lDAPI stostes to add in 5ml distilled water, mix, 2 μ g/ml.
Example 3：The use of detection kit
1. conventional xylene dewaxing is carried out to required detection sample slice, graded ethanol aquation, sodium bisulfite is handled,
Protease digestion, HCl immersions, graded ethanol dehydration, acetone fixes, 56 DEG C of roasting pieces 5 minutes, plus 10 μ l probe face liquid are in tissue
In section, 73 DEG C of denaturation 5 minutes in situ hybridization instrument after hybridizing, 42 DEG C of wet box hybridized overnights 16 hours, 50% formamide, lemon
Lemon acid buffer, 0.1%NP-40 and the rinsing of 70% ethanol, dark place spontaneously dry slide, and DAPI is redyed, mounting.Place 20 in dark place
In fluorescence microscopy Microscopic observation after minute.
2. result judgement：According to the marker color of different probe, respectively 30 cells of numeration, count Ratio values (Ratio=
Green cell number in the nucleus of red blood cell number/30 in 30 nucleus).
Ratio<1.8 be negative findings：Show that the sample is expanded without related gene.
Ratio>2.2 be positive findings：Show that related gene is expanded in sample.
Ratio represents that low-level is expanded between 2.3 and 4.0, and level is expanded in 4.1 to 10 expressions, represents high more than 10
Level is expanded.
Fig. 1 shows breast cancer lesion histotomy fluorescence in situ hybridization detection result, and the blue-fluorescence detected is ER
Probe develops the color, and purple develops the color for Ki-67 probes.
Obtained result is combined to different probe and carries out comprehensive analysis, and corresponding diagnosis is provided under physician guidance and is tied
Really, and to related prognosis judge.
Detection all carries out Quality Control piece operation, Quality Control piece and the biconditional operation of case sample one simultaneously every time.Quality Control piece can be bought
Commercialization is voluntarily prepared to photo or the known check sample of selection.
Above content is to combine specific/preferred embodiment made for the present invention be further described, it is impossible to
Assert that the specific implementation of the present invention is confined to these explanations.Come for general technical staff of the technical field of the invention
Say, without departing from the inventive concept of the premise, it can also make some replacements or modification to the embodiment that these have been described,
And these are substituted or variant should all be considered as belonging to protection scope of the present invention.
Sequence table
<110>Bioisystech Co., Ltd of hundred million cubes of Shenzhen
<120>Molecular probe and detection kit for breast cancer fluorescence in situ hybridization detection
<130> 17A100305JYC
<160> 5
<210> 1
<211> 145
<212> DNA
<213>Artificial sequence
<400> 1
acacggtgcg cgaggccggc ccgccggcat tctacaggcc aaattcagat aatcgacgcc 60
agggtggcag agaaagattg gccagtacca atgacaaggg aagtatggct atggaatctg 120
ccaaggagac tcgctactgt gcagt 145
<210> 2
<211> 123
<212> DNA
<213>Artificial sequence
<400> 2
actgctgtgt cgcccagccg caggtccgtt cccggggagc cagacctcgg acaccttgcc 60
tgaagtttcg gccataccta tctccctgga cgggctactc ttccctcggc cctgccaggg 120
aca 123
<210> 3
<211> 130
<212> DNA
<213>Artificial sequence
<400> 3
gaggtgcagg gctacgtgct catcgctcac aaccaagtga ggcaggtccc actgcagagg 60
ctgcggattg tgcgaggcac ccagctcttt gaggacaact atgccctggc cgtgctagac 120
aatggagacc 130
<210> 4
<211> 130
<212> DNA
<213>Artificial sequence
<400> 4
ctccaaagac ttcatatgct ccagatctcc tacttccgcg acccctatca cgtgtggtac 60
cagggcaacg cgtcgctggg gggacaccta acgcacgtgc tggaaggccc agacaccaac 120
accacgatca 130
<210> 5
<211> 145
<212> DNA
<213>Artificial sequence
<400> 5
tagtcctagg aaaactccag ttgccagtga tcaacgccgt aggtcctgca aaacagcccc 60
tgcttccagc agcaaatctc agacagaggt tcctaagaga ggagggagaa agagtggcaa 120
cctgccttca aagagagtgt ctatc 145
Claims (8)
1. a kind of molecular probe for breast cancer fluorescence in situ hybridization detection, it is characterised in that including one in following probe
Plant or a variety of：
Seq ID NO in ER probes, its sequence such as sequence table:Shown in 1 sequence；
Seq ID NO in PR probes, its sequence such as sequence table:Shown in 2 sequences；
Seq ID NO in HER-2 probes, its sequence such as sequence table:Shown in 3 sequences；
Seq ID NO in EPCR probes, its sequence such as sequence table:Shown in 4 sequences；
Seq ID NO in Ki-67 probes, its sequence such as sequence table:Shown in 5 sequences.
2. a kind of detection kit of the molecular probe comprising described in claim 1, it is characterised in that it has hybridization solution
(X2), denaturing liquid and DAPI counterstains.
3. detection kit according to claim 2, it is characterised in that the hybridization solution contains five kinds of specific probes, point
It is not：Seq ID NO in ER probes, its sequence such as sequence table:Shown in 1：Seq ID in PR probes, its sequence such as sequence table
NO:Shown in 2：Seq ID NO in HER-2 probes, its sequence such as sequence table:Shown in 3：EPCR probes, such as sequence table of its sequence
In Seq ID NO:Shown in 4：Seq ID NO in Ki-67 probes, its sequence such as sequence table:Shown in 5.
4. detection kit according to claim 3, it is characterised in that the hybridization solution is divided into two pipes, often pipe is containing
Three kinds of probes in five kinds of specific probes are stated, wherein EPCR contains in two pipes, other probes only contain in a pipe.
5. detection kit according to claim 4, it is characterised in that the hybridization solution is included：10% (w/v) sulfuric acid Portugal
Glycan, 10mM NaCl, 20% (v/v) formamide, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) PVP,
5mM Na2EDTA, 0.1% (v/v) TritonX-100,50mM Tris/HCl (pH7.5)；And respectively 30pM fluorescence mark
Five kinds of specific probes of note.
6. the detection kit according to any one of claim 2 to 5, it is characterised in that the DAPI counterstains it is main
Composition is DAPI (4,6- diamidino -2- benzene indole hydrochloride) and anti-colour fading liquid.
7. detection kit according to claim 6, it is characterised in that DAPI (4,6- diamidino -2- benzene indole hydrochloride)
Concentration be 4mg/ml.
8. the detection kit according to any one of claim 2 to 7, it is characterised in that also including separating and concentrating packaging
The packing box of reagent bottle or pipe.
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