CN108342481A - One group of probe and related kit for being directed to HER2 genes - Google Patents
One group of probe and related kit for being directed to HER2 genes Download PDFInfo
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- CN108342481A CN108342481A CN201810228031.0A CN201810228031A CN108342481A CN 108342481 A CN108342481 A CN 108342481A CN 201810228031 A CN201810228031 A CN 201810228031A CN 108342481 A CN108342481 A CN 108342481A
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- 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
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Abstract
The invention discloses probes and related kit that one group is directed to HER2 genes.By ERBB2 1 to ERBB2 20, this 20 kinds of probes form the one group provided by the invention probe for HER2 genes；The sequence of ERBB2 1 to ERBB2 20 is respectively sequence 1 20 in sequence table, and each probe is marked by fluorescent material.Experiments have shown that, using the present invention the probe for HER2 genes can pass flag into the cell or histotomy ErbB-2 (HER2) gene, can conveniently, accurately and rapidly in marked tumor cell or non-tumor cell tumor-marker gene HER2 expression and distribution, occurrence and development stage and the trend that can be used for assisting to judge tumour；The stable bond power and specificity of the probe of the present invention are high, and unspecific background signal is low, has very high application prospect.
Description
Technical field
The present invention relates in biotechnology, one group of probe and related kit for being directed to HER2 genes.
Background technology
(HER2) gene of human epidermal growth factor acceptor -2 also known as ERBB2, c-erbB-2, EGF-R ELISA
(EGFR) family member belongs to proto-oncogene, is located at 17q21, the transmembrane glycoprotein (cross-film of coding relative molecular mass 185KD
Tyrosine kinase receptor)；Research shows that：There are amplification/overexpressions of HER2 genes (such as breast in 30% or more human tumor
Gland cancer, oophoroma, carcinoma of endometrium, carcinoma of fallopian tube, gastric cancer and prostate cancer etc.)；HER2 is also that oophoroma, breast cancer etc. are important
The Index for diagnosis factor, the oophoromas of the HER2 positives, breast cancer are tumor-infiltrated to conventional chemotherapy and endocrine therapy Low Response
Property is strong, and DFS phase is short, poor prognosis.The breast cancer of HER2 positive (be overexpressed or expand), clinical characters and biological scholarship and moral conduct
Also to there are Special Manifestations, treatment mode also to be made a big difference with other kinds of breast cancer.Therefore, quick and precisely differentiate HER2
Assaypositive tissue cell treats important in inhibiting for the diagnosis of clinical case and effective medication.
HER2 is generally detected using immunohistochemistry (IHC) about Epidermal growth factor-recepor-2 (HER2) detection at present
Protein overexpression detects the level of HER2 gene magnifications using hybridization in situ.The detection mode that FDA has been approved by includes exempting from
Epidemic disease group (IHC), fluorescence in-situ hybridization method (fluorescence in situ hybridization, FISH) and colour developing are former
Position hybridizing method (chromogenic in situ hybridization, CISH).In addition there are bright visual field in situ hybridizations
(brightfield in situ hybridization).Due to CISH have compared with FISH coincidence rate is high, observation is convenient,
Sample can long-term preservation the features such as, used by many testing agencies both at home and abroad.But different detection method, calculating it is accurate
Property and reagent selection can all influence HER2 detection result.One investigation result in the U.S. shows that nearly 1/4 patient is because of detection
As a result inaccurate and receive inappropriate treatment.IHC detects the error rate average out to 18% of HER2 protein overexpressions, FISH
The error rate of HER2 gene magnifications is detected 13%.But it is usually necessary to use be with Her2 genes during preparing probe at present
Template, in Her2 genes non repetitive sequence carry out polymerase chain reaction, amplified production usually hundreds of base-pair sizes not
Deng DNA fragmentation, product obtains Her2 gene order fluorescence in situ hybridization probe after fluorescent marker, glimmering for Her2 genes
Light in situ hybridization detects, and in the process, the selection of segment designs, and PCR amplification efficiency, mutation introduces, and fluorescent label efficiency is non-
The problems such as specific binding, reaction cost, may all influence the use and popularization of Related product.In addition longer probe is for target
The recognition capability of sequence variations decreases again.Especially for being only single base (SNP) or different two sequences of a small number of base,
Cloning probe cannot distinguish between, and often hybridization signal is suitable, easily lead to the appearance of false positive.
Invention content
It is with fluorescence signal and thin for marking with modification of nucleic acids sequence by several that the object of the present invention is to provide a kind of
The complete probe of intracellular human epidermal growth factor acceptor -2 (HER2) gene nucleic acid.
Complete probe provided by the invention, includes the probe of entitled ERBB2-1, ERBB2-16 and ERBB2-18；It is described
The sequence of ERBB2-1, the ERBB2-16 and the ERBB2-18 are respectively sequence 1,16 and 18 in sequence table.
The complete probe can be only made of ERBB2-1, ERBB2-16 and ERBB2-18.Described in the complete probe
The molal quantity of ERBB2-1, the ERBB2-16 and the ERBB2-18 can be equal.
In above-mentioned probe, the complete probe can also be respectively by ERBB2-1, ERBB2-16 and ERBB2-18 and title
ERBB2-2、ERBB2-3、ERBB2-4、ERBB2-5、ERBB2-6、ERBB2-7、ERBB2-8、ERBB2-9、ERBB2-10、
ERBB2-11, ERBB2-12, ERBB2-13, ERBB2-14, ERBB2-15, ERBB2-17, ERBB2-19 and ERBB2-20 this 17
At least one of kind probe composition；
It is the ERBB2-2, the ERBB2-3, the ERBB2-4, the ERBB2-5, the ERBB2-6, described
It is ERBB2-7, the ERBB2-8, the ERBB2-9, the ERBB2-10, the ERBB2-11, described
It is ERBB2-12, the ERBB2-13, the ERBB2-14, the ERBB2-15, the ERBB2-17, described
The sequence of ERBB2-19 and the ERBB2-20 be respectively sequence 2 in sequence table, 3,4,5,6,7,8,9,10,11,12,13,14,
15,17,19 and 20.
Each probe can be marked by fluorescent material in above-mentioned complete probe.The fluorescent material concretely CY3.
In above-mentioned complete probe, the molal quantity of the ERBB2-1~ERBB2-20 is equal.
When the complete probe is made of 20 probes, the molal quantity of each probe is equal in the reagent set.Institute
When stating complete probe and being made of 4-20 probe, it is satisfied by between probe included in the complete probe when being made of 20 probes
Complete probe in molar ratio relationship between correspondent probe.
In the complete probe each probe can independent packaging, can also be packaged together.
The present invention also provides the systems of detection HER2, and the system comprises the complete probes.
Above system, which may also include, utilizes the reagent and/or instrument needed for fluorescence in-situ hybridization method detection HER2.
The system can be the kit for only including reagent.
The detection HER2 can be the HER2 genes in detection tissue, cell or cell culture fluid.The detection HER2 can
To detect the expression and/or amplification of HER2 genes.The amplification refers to number of copies or expression quantity appearance in target gene
Increase.
The present invention also provides the complete probes or the system in the expression and/or distribution of label HER2 genes
Using.
The present invention also provides the complete probes or the system in the expression and/or distribution for preparing label HER2 genes
Application in product.
The present invention also provides the complete probes or the system to identify or assist identification tumour to occur and/or develop
In application.
The present invention also provides the complete probe or the system prepare identification or auxiliary identification tumour occur and/or
Develop the application in product.
The tumour can be entity tumor.The entity tumor can be breast cancer, oophoroma, carcinoma of endometrium, fallopian tubal
Cancer, gastric cancer or prostate cancer.
It is demonstrated experimentally that using the present invention complete probe can pass flag is intracellular or histotomy human epidermal growth because
Sub- receptor -2 (HER2) gene, can conveniently, accurately and rapidly tumor-marker gene in marked tumor cell or non-tumor cell
The expression and distribution of HER2, occurrence and development stage and the trend that can be used for assisting to judge tumour.The present invention complete probe it is steady
Determine binding force and specificity is high, unspecific background signal is low, has very high application prospect.
Description of the drawings
Fig. 1 is the result for the probe for targeting ACTB genes.Wherein, ACTB indicate targeting ACTB genes probe as a result,
DAPI indicates that dyeing of the DAPI to nucleus, MERGE indicate the merging of preceding two width figure.
Fig. 2 is the result of NC probe in detecting MCF7 and 293T.
Fig. 3 is the testing result of HER2 genes in three kinds of cells.HER2+17 indicates No. 17 dyes of complete probe 2 and targeting
The probe in colour solid kinetochore region detects together, when detecting the molal quantity all same of every probe used.
Fig. 4 is the testing result of human breast carcinoma tissue paraffin section.
Fig. 5 is the testing result of human breast carcinoma tissue frozen section.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Agents useful for same is as follows：
20 × SSC is the sterile solution being made of solvent and solute, and solvent is DEPC water, and solute and its concentration are respectively
NaCl 175.3g/L, monohydrate potassium sodium 63g/L, pH 7.0；2 × SSC and 0.4 × SSC is respectively by sterile DEPC water by 20
× SSC dilutes 10 times and 50 times and obtains, three kinds of equal room temperature preservations of reagent.
Hybridization buffer is the sterile solution being made of solvent and solute, and solvent is DEPC water, solute and its concentration difference
For NaCl 5.265g/100ml, deionized formamide 60ml/100ml, 20 × SSC 25ml/100ml, 10% (quality percentage
Than) SDS aqueous solution 5ml/100ml, dextran sulfate 10g/100ml, room temperature preservation is spare.
0.1%tritonX-100 is the tritonX-100 concentration expressed in percentage by volumes added tritonX-100 into PBS and obtained
For 0.1% solution, room temperature preservation is spare.
Proteinase K Solution is using 2 × SSC by Proteinase K according to 1:1000 dilutions obtain, -20 DEG C of preservations.
0.4 × SSC/0.3%Tween20 is that the Tween20 volume basis that addition Tween20 is obtained into 0.4 × SSC is dense
The solution that degree is 0.3%.
2 × SSC/0.1%Tween20 is to add the Tween20 concentration expressed in percentage by volumes that Tween20 is obtained into 2 × SSC to be
0.1% solution.
Denaturing liquid is the solution being made of solvent and solute, solvent ddH2O, solute and its concentration is respectively 20 ×
SSC4ml/40ml, deionized formamide：28ml/40ml, each matching while using.
Rinsing solution is the solution being made of solvent and solute, solvent ddH after hybridization2O, solute and its concentration are respectively
20 × SSC 4ml/40ml, deionized formamide 20ml/40ml, each matching while using.
0.1mol citrate buffers are the solution being made of solvent and solute, solvent ddH2O (ultra-pure water), solute and
Its concentration is respectively citric acid trisodium 3g/L, citric acid 0.4g/L.
Probe should be protected from light dilution and preserve, and the operation being added in experimentation after probe should carry out under the conditions of being protected from light.
DAPI is with PBS according to 1:1000 dilutions, need to be kept in dark place and use.
The preparation of the complete probe of embodiment 1, detection HER2 assaypositive tissues or cell
It is provided in this embodiment detection HER2 assaypositive tissues or cell complete probe be complete probe 1 and complete probe 2,
Complete probe 1 is made of three probes that title is respectively ERBB2-1, ERBB2-16 and ERBB2-18, and complete probe 2 is by title
Respectively the 20 of ERBB2-1~ERBB2-20 probe compositions.Each probe sequence is as shown in table 1, the ends each probe 5' mark have
CY3。
Table 1, detecting probe information
| Probe title | Probe sequence |
| ERBB2-1 | 5'GCTCCTTGGTCCTTCACCTAACCTTGCCC 3'(sequences 1) |
| ERBB2-2 | 5'CGTCAATGTCCAGCAGCCGAGCCA 3'(sequences 2) |
| ERBB2-3 | 5'TGTGATGCCAGGAGACAGACCCTACCCG 3'(sequences 3) |
| ERBB2-4 | 5'GCAGGTCGGGCAGCAGGCAGAGG 3'(sequences 4) |
| ERBB2-5 | 5'CCTCCCAACCATCACAAACCACAGCCA 3'(sequences 5) |
| ERBB2-6 | 5'GCAGGTCGGGCAGCAGGCAGAGG 3'(sequences 6) |
| ERBB2-7 | 5'GCCCAGTCCATAAACGCCCATCAGTA 3'(sequences 7) |
| ERBB2-8 | 5'GCACAGGGCTTGCTGCACTTCTCA 3'(sequences 8) |
| ERBB2-9 | 5'GGATACACTGCCACTTGTTTATTCCTTCACCT 3'(sequences 9) |
| ERBB2-10 | 5'GTCCCAAGAGGGTCTGAGGAAGGATAGG 3'(sequences 10) |
| ERBB2-11 | 5'GGGAGGCTGAGGTGGGCGGAT 3'(sequences 11) |
| ERBB2-12 | 5'GTGGTATTGTTCAGCGGGTCTCCATTGTCT 3'(sequences 12) |
| ERBB2-13 | 5'CACTGGGTTGTAAGTTGGGAGTTTGCGG 3'(sequences 13) |
| ERBB2-14 | 5'TGGCGGGCAGGCACTGGGTTGTA 3'(sequences 14) |
| ERBB2-15 | 5'GGGTAGAGCACATTGGGCACAAAGCAGAG 3'(sequences 15) |
| ERBB2-16 | 5'TTGTTCAGCGGGTCTCCATTGTCT 3'(sequences 16) |
| ERBB2-17 | 5'CTGGGCTGAAGGCAGGAGGAGGGTGG 3'(sequences 17) |
| ERBB2-18 | 5'GGGTGGTATTGTTCAGCGGGTCTCCATTGTCT 3'(sequences 18) |
| ERBB2-19 | 5'TGCTTGGGAAGTAGAGGGATTTCAGGGG 3'(sequences 19) |
| ERBB2-20 | 5'TGGATGGAAAGGTCATTCGCCTGAACAAG 3'(sequences 20) |
The molar ratio of ERBB2-1, ERBB2-16 and ERBB2-18 are 1 in complete probe 1：1：1, in complete probe 2
The molal quantity of ERBB2-1~ERBB2-20 is equal.Each equal independent packaging of probe in complete probe 1 and complete probe 2.
The probe (table 2) in No. 17 kinetochore regions of design targeting and targeting are used as reference gene ACTB genes
(Homo sapiens actin beta(ACTB),mRNA NCBI Reference Sequence:
NM_001101.4 probe (table 3)), and design NC probes (5'-GTGTAACACGTCTATACGCCCA-3') work
It for negative control, targets the molal quantity in the probe in No. 17 kinetochore regions between each probe and is equal, in targeting conduct
Join the molal quantity in the probe of Gene A CTB genes between each probe to be equal.
Table 2, the probe for targeting No. 17 kinetochore regions
| Probe title | Probe sequence |
| CHR17-1 | The bis- marks of 5'TGAACTCGCAGAGGTGAACATTCCT 3'(FAM) |
| CHR17-2 | The bis- marks of 5'TCTCAGAGCCCTCTTCGTGGTGTT 3'(FAM) |
| CHR17-3 | The bis- marks of 5'GAACATTCCGTTGGATGGAGCAGT 3'(FAM) |
| CHR17-4 | The bis- marks of 5'CTCCGAAGATGTCTTTGGAAACGG 3'(FAM) |
| CHR17-5 | The bis- marks of 5'TTGGACCGCTCTGAGGATTTCGT 3'(FAM) |
| CHR17-6 | The bis- marks of 5'CAGAGCTGAACACTCCTTGCGATGTA 3'(FAM) |
| CHR17-7 | The bis- marks of 5'AACGGGATAAACTGCACAGAACTAAACAG 3'(FAM) |
| CHR17-8 | The bis- marks of 5'TGGAAACGCGATAACTGCACCTAA 3'(FAM) |
| CHR17-9 | The bis- marks of 5'TGGAAACGGGATAAACCGCACA 3'(FAM) |
| CHR17-10 | The bis- marks of 5'TTCTGTGGCATCCGCAAGGG 3'(FAM) |
| CHR17-11 | The bis- marks of 5'GGGAGGCTGAGGTGGGCGGAT 3'(FAM) |
| CHR17-12 | The bis- marks of 5'TCTGAGGAATTTGTTGGAAACGGG 3'(FAM) |
| CHR17-13 | The bis- marks of 5'GAGCTGAGCATTCCTTGCGATGTA 3'(FAM) |
| CHR17-14 | The bis- marks of 5'TGGAAACGGGATAAACCGCACA 3'(FAM) |
| CHR17-15 | The bis- marks of 5'TTTGGAGGGCTTTGTGGTTTGTG 3'(FAM) |
| CHR17-16 | The bis- marks of 5'GGATAAACCGCACAGAACTAAACAGAAGA 3'(FAM) |
| CHR17-17 | The bis- marks of 5'GGAATCTGCAAGTGGATATTTGGGC 3'(FAM) |
| CHR17-18 | The bis- marks of 5'CGTTTGGAGGGCTTTGTGGTTTG 3'(FAM) |
| CHR17-19 | The bis- marks of 5'AACGGGATAACTGCACCTAACTAAACGG 3'(FAM) |
| CHR17-20 | The bis- marks of 5'GGATAAACCGCACAGAACTAAACAGAAGA 3'(FAM) |
Table 3, the probe for targeting ACTB genes
Wherein, the bis- marks expressions of FAM mark at the both ends of corresponding sequence and have in table 2 and table 3.
The detection of embodiment 2, HER2 assaypositive tissues or cell
The present embodiment detects the HER2 positives respectively using the detection HER2 assaypositive tissues of embodiment 1 or the complete probe of cell
Tissue or cell.
One, in attached cell or suspension cell (or suspended components) the HER2 positives detection
(1) attached cell (by taking 48 orifice plates as an example)
Sample to be tested is breast cancer cell MCF7, ovarian cancer cell SKOV3 and control cell 293T.Specific detection method is such as
Under：
A) processed and sizeable coverslip is put into 48 orifice plates, according to 1 × 104The density of cells/well
Attached cell in culture medium is inoculated on the coverslip in 48 orifice plates, in 37 DEG C of 5%CO2It was cultivated in incubator
Night；
B) culture medium abandoned in orifice plate is inhaled, PBS is washed twice, each 5min；
C) it inhales and abandons PBS, 100ul absolute ethyl alcohols are added per hole, room temperature fixes 15min；
D) it inhales and abandons absolute ethyl alcohol, 100ul 0.1%tritonX-100 (matching while using) room temperature is added per hole and handles cell
15min；
E) it inhales and abandons 0.1%tritonX-100, PBS is washed twice, each 5min；
F) it inhales and abandons PBS, 2 × SSC of 100ul are added per hole, 37 DEG C of incubators place 30min；
G) it inhales and abandons 2 × SSC, 100ul 70% (percent by volume) ethanol water is added per hole, is placed at room temperature for 3min；
H) it inhales and abandons 70% ethanol water, 100ul 85% (percent by volume) ethanol water is added per hole, room temperature is put
Set 3min；
I) it inhales and abandons 85% ethanol water, 100ul absolute ethyl alcohol aqueous solutions are added per hole, are placed at room temperature for 3min；
J) it inhales and abandons absolute ethyl alcohol, drying at room temperature；
K) probe mixed liquor is prepared：
Hybridization buffer is incubated 2h in 37 DEG C of water-baths in advance；The 40ul DEPC water of complete probe 2 of 1OD embodiments 1
Dissolving obtains probe solution, and a concentration of 1ug/ul is protected from light spare；
100ul probe mixed liquors are prepared per hole：Probe solution, hybridization buffer 70ul, DEPC water complement to 100ul.It visits
A concentration of 50ug/ml, 20ug/ml, 12.5ug/ml, tetra- gradients of 6ug/ml are arranged in needle；73 DEG C of water-baths are denaturalized 5min；
L) 100ul probe mixed liquors are added per hole, are incubated overnight in 37 DEG C of incubators；
M) hybridize next day, sample is taken out from 37 DEG C of incubators, probe mixed liquor is abandoned in suction, and 100ul is added per hole in 65 DEG C
0.4 × SSC/0.3%Tween20 of preheating washs 2min；
N) it inhales and abandons 0.4 × SSC/0.3%Tween20,2 × SSC/0.1%Tween20 100ul of 100ul are added per hole,
Room temperature washing 2min, cleaning solution is abandoned in suction, in drying at room temperature；
O) the DAPI dye liquors after 100ul dilutions are added per hole, are protected from light dyeing 20min, dye liquor is abandoned in suction, and 100ul PBS are added,
Choose coverslip, under the microscope in fluorescence microscopy.
According to the method described above, the complete probe 2 of embodiment 1 is replaced with to the spy in No. 17 kinetochore regions of targeting
(there are two types of NC probes, a kind of to be marked with FAM at 5 ' ends, is denoted as NC-FAM for needle, the probe for targeting ACTB genes and NC probes；One
Kind is marked with CY3 at 5 ' ends, is denoted as NC-CY3, is tested respectively), the knot under fluorescence microscopy microscopic observation different probe
Fruit.
Breast cancer cell MCF7, ovarian cancer cell SKOV3 and control cell 293T are at the probe through targeting ACTB genes
After reason, equal visible green fluorescence signal (Fig. 1).The results are shown in Figure 2 by two kinds of NC probe in detecting MCF7 and 293T, same
It is displayed without in operating process and apparent non-specific fluorescence combination occurs.
The results are shown in Figure 3 for the probe in No. 17 kinetochore regions of complete probe 2 and targeting, the results show that newborn
There is gene copy quantity increase in nucleus in ERBB2 (HER2) gene in adenocarcinoma cell MCF7/ ovarian cancer cell SKOV3s, carefully
ERBB2 (HER2) gene expression amount increases in born of the same parents' endochylema, occurs that multiple spot is more or larger fluorescence cluster；Relative comparison cell
293T carries out destination gene expression level and No. 17 kinetochore repetitive sequence design probe results are shown in endochylema and core
Expression is in normal level, meets MCF7 and SKOV3 cell HER2 gene masculine features.And now existing research shows that
HER2 genes are respectively MCF7 and SKOV3 cells in breast cancer cell MCF7, ovarian cancer cell SKOV3 and control cell 293T
HER2 gene masculine features, and it is normally to have 2 fluorescence punctuates in chromosome double-strand that 293T cells, which are presented negative,.The present embodiment
In result it is consistent with the result of existing research.
(2) suspension cell or suspended components
Sample to be tested is the cell suspending liquid of breast cancer cell MCF7, ovarian cancer cell SKOV3 and control cell 293T.Tool
Body detecting method is as follows：
A) suspension cell (ingredient) is fixed on slide：
Method one：The suspension cell for being fixed on absolute ethyl alcohol is set to be attached on slide (poly-D-lysine processing) with rejection tablet machine；
Method two：Cell suspension is dropped on the processed glass slide of poly-D-lysine, a piece of glass slide is separately taken to do by smear
Push jack is moved right from cell drop left side, is put down to the left when cell drop is uniformly adhered between two panels, then by push jack by push jack
It quietly moves (two panels is at 30-40 degree angle) and releases uniform cell membrane, in being dried after several times on alcolhol burner.
B) cell smear dried is placed in 10cm culture dishes, 0.1%tritonX-100 is added dropwise and digests 15min；
C) it inhales and abandons 0.1%tritonX-100, PBS is added dropwise and washes twice, each 5min；
D) it inhales and abandons PBS, 2 × SSC of 100ul are added dropwise, 37 DEG C of incubators place 30min；
E) it inhales and abandons 2 × SSC, 100ul 70% (percent by volume) ethanol water is added dropwise, is placed at room temperature for 3min；
F) it inhales and abandons 70% ethanol water, 100ul 85% (percent by volume) ethanol water is added dropwise, is placed at room temperature for
3min；
G) it inhales and abandons 85% ethanol water, 100ul absolute ethyl alcohols are added dropwise, are placed at room temperature for 3min；
H) it inhales and abandons absolute ethyl alcohol, drying at room temperature；
I) probe mixed liquor is prepared：
Hybridization buffer is incubated 2h in 37 DEG C of water-baths in advance；The 40ul DEPC water of complete probe 1 of 1OD embodiments 1
Dissolving obtains probe solution, and a concentration of 1ug/ul is protected from light spare；
100ul probe mixed liquors are prepared per hole：Probe solution, hybridization buffer 70ul, DEPC water complement to 100ul.It visits
A concentration of 50ug/ml, 20ug/ml, 12.5ug/ml, tetra- gradients of 6ug/ml are arranged in needle；
75-80 DEG C of water-bath is denaturalized 5min；
J) 100ul probe mixed liquors are added dropwise in every slide, and covered is placed in wet box, and 37 DEG C of incubators were incubated
Night；
K) hybridize next day, sample taken out from 37 DEG C of incubators, probe mixed liquor is abandoned in suction, every slide dropwise addition 100ul in
0.4 × SSC/0.3%Tween20 of 65 DEG C of preheatings washs 2min；
L) it inhales and abandons 0.4 × SSC/0.3%Tween20,2 × SSC/0.1% of 100ul is added dropwise in every slide
Tween20100ul, room temperature washing 2min, cleaning solution is abandoned in suction, in drying at room temperature；
M) the DAPI dye liquors after 100ul dilutions are added dropwise in every slide, are protected from light dyeing 20min, and dye liquor, every slide drop are abandoned in suction
Add 100ul PBS to wash 2 times, glycerine covered is added dropwise, under the microscope in fluorescence microscopy.
According to the method described above, the complete probe 1 of embodiment 1 is replaced with to No. 17 kinetochore regions of targeting respectively
Probe, the probe and NC probes for targeting ACTB genes, the result under fluorescence microscopy microscopic observation different probe.
The results show that the result of step (2) is consistent with the result of step (1).
(3) quantitative PCR
Utilize the primers F of HER2：CCTGCTGAACTGGTGTATGC and R：GCCCGAAGTCTGTAATTTTGA is respectively to breast
Adenocarcinoma cell MCF7, ovarian cancer cell SKOV3 and control cell 293T carry out quantitative fluorescent PCR, and the PCR product of the primer pair is
134bp.Using ACTB as internal reference, internal control primer sequence is F：CTCCCTGGAGAAGAGCTACGAGC and R：
CCAGGAAGGAAGGCTGGAAGAG.The results show that HER2 genes are in breast cancer cell MCF7, ovarian cancer cell SKOV3 and right
Photo cell 293T is respectively that MCF7 and SKOV3 cells are in HER2 gene masculine features, and HER2 gene negatives are presented in 293T cells
Feature, the result obtained using the complete probe 1 and 2 of the present invention are consistent with the above results.
Two, the detection of the histotomy HER2 positives
1, paraffin section
Sample to be tested is human breast carcinoma tissue paraffin section (have passed through research object informed consent).Specific detection method is such as
Under：
A. it dewaxes：
(a) paraffin section preheats 30min in 60 DEG C of ovens；
(b) 60 DEG C of placement 20min in dimethylbenzene 100ul are added dropwise in every slice；
(c) (100%, 95%, 90%, 80%) is incubated at room temperature each 10min in graded ethanol aqueous solution.
B. Protease Treatment：
(a) Proteinase K Solution is preheated to 37 DEG C；
(b) Proteinase K Solution 100ul, 37 DEG C of incubation 20min is added dropwise in every slice；
(c) every slice is added dropwise 2 × SSC 100ul and washes slice at room temperature 3 times, each 1min；
(d) graded ethanol aqueous solution (70%, 80%, 90%, 100% (- 20 DEG C of precoolings)) dehydration, each 2min, air
Middle drying；
C. it is denaturalized：
(a) 75-80 DEG C of preheating denaturing liquid；
(b) preheating denaturing liquid 100ul, 78 DEG C of incubation 8min is added dropwise in every slice；
(c) graded ethanol aqueous solution (70%, 80%, 90%, 100% (- 20 DEG C of precoolings)) dehydration, each 2min, air
Middle drying；
D. hybridize：
(a) prepare probe mixed liquor：The complete probe 1 of 1OD embodiments 1 obtains probe solution with 40ul DEPC water dissolutions,
A concentration of 1ug/ul is protected from light spare；
With manufacturing probe mixed liquor, contain per 100ul probe mixed liquors：Probe solution, hybridization buffer 70ul, DEPC water are supplied
To 100ul.A concentration of 50ug/ml, 20ug/ml, 12.5ug/ml, tetra- gradients of 6ug/ml are arranged in probe；73 DEG C of water-bath denaturation
3-8min；
(b) prepare wet box, intersect and place slice；
(c) drop 100ul probe mixed liquors are capped slide on slice；
(d) wet box lid, 37 DEG C of incubation 12-16h are covered.
E. it is washed after hybridizing：
(a) rinsing solution after 43 DEG C of preheating hybridization；
(b) tweezers carefully remove the coverslip on slice, and rinsing solution 100ul is washed after the hybridization of preheating is added dropwise in every slice
It is sliced 15min；
(d) 2 × SSC (being preheated to 37 DEG C) is washed 2 times, each 10min；
(e) PBS washes 1 time, 10min.
F. nuclear targeting：
(a) every slice plus 10ul DAPI cover coverslip and are protected from light incubation 5min at room temperature；
(b) microscopically observation, or it is placed in 4 DEG C of preservations in wet box.
According to the method described above, the complete probe 1 of embodiment 1 is replaced with into NC probes (being marked with FAM at 5 ' ends), in fluorescence
Result under microscopically observation different probe.
The results show that occur apparent positive signal (left figure in Fig. 4) in the testing result of complete probe 1, and NC is visited
Needle operates no apparent positive signal (right figure in Fig. 4) in the same terms.
2, frozen section
Sample to be tested is human breast carcinoma tissue frozen section (have passed through research object informed consent).Specific detection method is such as
Under：
1. rehydration：
1) frozen section takes out, and 0.1mol citrate buffer 100ul are added dropwise in every slice, are placed at room temperature for 15min；
2) it inhales and abandons 0.1mol citrate buffers, PBS 100ul are added dropwise and wash twice, each 5min；
2. Protease Treatment：
1) Proteinase K Solution is preheated to 37 DEG C；
2) Proteinase K Solution 100ul, 37 DEG C of incubation 20min is added dropwise in every slice；
3) every slice is added dropwise 2 × SSC 100ul and rinses slice at room temperature 3 times, each 1min；
4) graded ethanol aqueous solution (70%, 80%, 90%, 100% (- 20 DEG C of precoolings)) is dehydrated, each 2min, in air
It is dry.
3. denaturation：
1) 78 DEG C of preheating denaturing liquids；
2) the denaturing liquid 100ul after preheating is added dropwise in every slice, is incubated 8min；
3) graded ethanol aqueous solution (70%, 80%, 90%, 100% (- 20 DEG C of precoolings)) is dehydrated, each 2min, in air
It is dry.
4. hybridization：
1) prepare probe mixed liquor：The complete probe 2 of 1OD embodiments 1 obtains probe solution with 40ul DEPC water dissolutions,
A concentration of 1ug/ul is protected from light spare；
With manufacturing probe mixed liquor, contain per 100ul probe mixed liquors：Probe solution, hybridization buffer 70ul, DEPC water are supplied
To 100ul.A concentration of 50ug/ml, 20ug/ml, 12.5ug/ml, tetra- gradients of 6ug/ml are arranged in probe；73 DEG C of water-bath denaturation
5min；
2) prepare wet box, intersect and place slice；
3) drop 100ul probe mixed liquors are capped slide on slice；
4) wet box lid, 37 DEG C of incubation 12-16h are covered.
5. being washed after hybridization：
1) rinsing solution after 43 DEG C of preheating hybridization；
2) tweezers carefully remove the coverslip on slice, and rinsing solution 100ul43 after the hybridization of preheating is added dropwise in every slice
DEG C wash slice 15min；
3) 2 × SSC (being preheated to 37 DEG C) is washed 2 times, each 10min；
4) PBS washes 1 time, 10min.
6. nuclear targeting：
1) every slice plus 10ulDAPI cover coverslip and are protected from light incubation 20min at room temperature；
2) microscopically observation, or it is placed in 4 DEG C of preservations in wet box.
According to the method described above, the complete probe 2 of embodiment 1 is replaced with to No. 17 kinetochore regions of targeting respectively
Probe, the probe for targeting ACTB genes and NC probes (being marked with FAM at 5 ' ends), under fluorescence microscopy microscopic observation different probe
Result.
The results show that occur apparent positive signal (left figure in Fig. 5) in the testing result of complete probe 2, and NC is visited
Needle operates no apparent positive signal (right figure in Fig. 5) in the same terms.
<110>Suzhou GenePharma Co., Ltd., Shanghai JiMa pharmacy Technology Co., Ltd
<120>One group of probe and related kit for being directed to HER2 genes
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 1
gctccttggt ccttcaccta accttgccc 29
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
cgtcaatgtc cagcagccga gcca 24
<210> 3
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
tgtgatgcca ggagacagac cctacccg 28
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
gcaggtcggg cagcaggcag agg 23
<210> 5
<211> 27
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
cctcccaacc atcacaaacc acagcca 27
<210> 6
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
gcaggtcggg cagcaggcag agg 23
<210> 7
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
gcccagtcca taaacgccca tcagta 26
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
gcacagggct tgctgcactt ctca 24
<210> 9
<211> 32
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
ggatacactg ccacttgttt attccttcac ct 32
<210> 10
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
gtcccaagag ggtctgagga aggatagg 28
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
gggaggctga ggtgggcgga t 21
<210> 12
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
gtggtattgt tcagcgggtc tccattgtct 30
<210> 13
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
cactgggttg taagttggga gtttgcgg 28
<210> 14
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
tggcgggcag gcactgggtt gta 23
<210> 15
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
gggtagagca cattgggcac aaagcagag 29
<210> 16
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
ttgttcagcg ggtctccatt gtct 24
<210> 17
<211> 26
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
ctgggctgaa ggcaggagga gggtgg 26
<210> 18
<211> 32
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
gggtggtatt gttcagcggg tctccattgt ct 32
<210> 19
<211> 28
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
tgcttgggaa gtagagggat ttcagggg 28
<210> 20
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
tggatggaaa ggtcattcgc ctgaacaag 29
Claims (10)
1. complete probe includes the probe of entitled ERBB2-1, ERBB2-16 and ERBB2-18；It is the ERBB2-1, described
The sequence of ERBB2-16 and the ERBB2-18 are respectively sequence 1,16 and 18 in sequence table.
2. complete probe according to claim 1, it is characterised in that：The complete probe by ERBB2-1, ERBB2-16 and
ERBB2-18 and title are respectively ERBB2-2, ERBB2-3, ERBB2-4, ERBB2-5, ERBB2-6, ERBB2-7, ERBB2-
8、ERBB2-9、ERBB2-10、ERBB2-11、ERBB2-12、ERBB2-13、ERBB2-14、ERBB2-15、ERBB2-17、
At least one of this 17 kinds of probes of ERBB2-19 and ERBB2-20 form；
The ERBB2-2, the ERBB2-3, the ERBB2-4, the ERBB2-5, the ERBB2-6, the ERBB2-7,
The ERBB2-8, the ERBB2-9, the ERBB2-10, the ERBB2-11, the ERBB2-12, the ERBB2-13,
The ERBB2-14, the ERBB2-15, the ERBB2-17, the ERBB2-19 and the ERBB2-20 sequence be respectively
Sequence 2,3,4,5,6,7,8,9,10,11,12,13,14,15,17,19 and 20 in sequence table.
3. complete probe according to claim 1 or 2, it is characterised in that：Each probe is by fluorescence in the complete probe
Mass signatures.
4. complete probe according to claim 2 or 3, it is characterised in that：The ERBB2-1~ERBB2-20's rubs
Your number is equal.
5. the system for detecting HER2, including any complete probe in claim 1-4.
6. system according to claim 5, it is characterised in that：The system also includes examined using fluorescence in-situ hybridization method
Survey the reagent and/or instrument needed for HER2.
7. any complete probe or the system of claim 5 or 6 are in the expression for marking HER2 genes in claim 1-4
And/or the application in distribution.
8. any complete probe or the system of claim 5 or 6 are preparing label HER2 genes in claim 1-4
Application in expression and/or distribution of products.
9. identification tumour is being identified or assisted to any complete probe or the system of claim 5 or 6 in claim 1-4
Generation and/or developing application.
10. any complete probe or the system of claim 5 or 6 are preparing identification or auxiliary mirror in claim 1-4
Determine tumour and occurs and/or develop the application in product.
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|---|---|---|---|
| CN201810228031.0A CN108342481A (en) | 2018-03-20 | 2018-03-20 | One group of probe and related kit for being directed to HER2 genes |
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