CN109022575A - A kind of molecular marker can be used for dog Diagnosis of Breast Tumor and Index for diagnosis - Google Patents
A kind of molecular marker can be used for dog Diagnosis of Breast Tumor and Index for diagnosis Download PDFInfo
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Abstract
The invention discloses the molecular marker that kind can be used for dog Diagnosis of Breast Tumor and Index for diagnosis, the molecular marker is ICAM1 gene, and nucleotide sequence is as shown in SEQ ID NO:1；And the RT-qPCR detection primer of a pair of of detection ICAM1 gene expression, nucleotide sequence is as shown in NO:2~3 SEQ ID.And the kit of a kind of dog Diagnosis of Breast Tumor and Index for diagnosis is prepared.The ICAM1 gene only significant low expression in malignant tumors group；From the ICAM1 gene expression amount in the dog breast tumor cell that transfer stove is isolated compared with primary tumor cell significant difference, illustrate that it can identify the good pernicious of dog tumor of breast and take part in the infiltration of dog tumor of breast, transfer process.Therefore, ICAM1 can be used as the molecular marker of dog Diagnosis of Breast Tumor and Index for diagnosis.Molecular marker relevant to the transfer of dog tumor of breast provided by the invention, sensibility and specificity with higher to the property identification of dog tumor of breast, prognosis evaluation, determine therapeutic scheme in time, improve the survival rate of illness dog only and have great importance.
Description
Technical field
The present invention relates to dog tumor of breast detection technique fields, can be used for dog tumor of breast more particularly, to one kind and examine
Disconnected and Index for diagnosis molecular marker.
Background technique
Dog tumor of breast is the highest tumor disease of bitch disease incidence, is mainly in 6~11 years old person in middle and old age's bitch, wherein
About 30%~50% is pernicious.Since environmental factor or genetic cause lead to occur lump in mammary gland, clinic is referred to as
For tumor of breast.A large number of studies show that hormone can be largely affected by the generation of dog tumor of breast, as estrogen, progestational hormone,
Growth hormone etc. will affect the generation of malignant tumour.The study found that dog suffers from the disease incidence of tumor of breast with heat time digital display
Work increases, and sterilization operation is carried out after first heat can effectively limit tumor of breast occurrence probability.
Society is developing, and times is changing moves, and pet dog comes into the family of common people, with the increase at dog age,
The probability for suffering from tumor of breast also slowly increases or even threat to life, and the pathogenesis for studying dog tumor of breast is extremely urgent, finds
Reliable and effective treatment method, to improve the survival rate of pet dog only and the existence time limit.In addition, pet dog is as companion animals,
It is co-located in identical living environment with the mankind, there is similar life habit, eating habit, the carcinogenic factor touched also has
General character, and the various biologicallies of dog tumor of breast, cell sign, antigenic type etc. are similar to people, therefore, study dog
Tumor of breast can not only be beneficial to veterinary clinic, also can provide some thinkings and foundation for mankind's medical oncology.
The maximum feature of malignant tumour is exactly infiltration and transfer, and the final treatment failure of tumor patient is caused to welcome death.
Grasping its complete mechanism of action is also the common target and wish of clinical practitioners.The infiltration metastasis of tumour is one complicated
Pathophysiological process.In this process, the transforming growth factor-β (transforming of normal stroma cell secretion
Growth factor- β, TGF-β) promote tumour cell that Epithelial and stromal Phenotypic Change (epithelial-mesenchymal occurs
Transition, EMT), so that tumour cell is lost the adhesive attraction and epithelial polarity of cell, the ability for being migrated and being invaded,
To promote tumor cell invasion blood vessel, final tumour cell enters blood circulation threat to life.
The tumour cell to fall off from primary tumor continues into the circulatory system of body, becomes circulating tumor cell (ci
Rculating tumor cells, CTCs), it is possible that developing into the transfer stove of distant place.Tumour cell can pass through change
Peritumoral tissues microenvironment and modify etc. approach to own face antigen to escape the monitoring of body immune system, identification
And attack, continue merisis, the i.e. immunologic escape of tumour, it is final that tumor cell migration to distal end target organ carries out infinite multiplication
Form transfer stove.
Currently, surgical resection is that pet clinically applies a kind of most methods.There is physical condition and spiritual shape
The dispirited dog of state implements to perform the operation again after need to waiting until stable disease；Bigger for the age or weaker constitution suffers from dog, then mostly not
It is proposed with the mode of surgical resection；If shifting, it is recommended that conservative therapy；But it is some suffer from dog thoroughly extract it is primary
There is the case where recurring and shifting again after tumour, often leads to suffer from dog death.Chemotherapy at present can only be as a kind of complementary
Treatment is used in the case where there is malignant tumour or the apparent situation of transfer.Currently, pet clinically chemotherapy apply compared with
More but traditional anti-tumor drugs plays drug effect and needs very high blood concentration, and the selectivity acted on is poor, is easy to normal
Histocyte generate lethal effect.Radiotherapy is that a kind of local therapeutic approaches of tumour are treated using radioactive ray, can be made
For a kind of auxiliary treatment means after the operation excision of essential dog tumor of breast.Targeted therapy is a kind for the treatment of in cellular level
Method, neoplasm targeted therapy means mainly include cell factor, adoptive cell, monoclonal antibody, gene therapy etc. at present.
The selection of the above various treatment methods is mostly to assess to suffer from tumor of breast dog only by the clinical experience of pet doctor
Prognosis situation, sensitivity is lower, for malignant tumour prognosis assessment still without reliable marker, therefore, find special
Property is strong, and the dog tumor of breast prognostic marker of high sensitivity is necessary.
It is cured clinically in people, due to the shortage of tumor prognosis marker, most breast cancer patients receive excessive
Chemotherapy has caused many side effects, including leukaemia, heart disease and severe infections.And a part need the patient of chemotherapy without
Method is given treatment in time, leads to tumour metastasis and recurrence, causes death.Therefore, suitable tumor prognosis marker will be to patient is judged
No to need to receive adjuvant treatment, improving patient's survival rate has great significance.
Pet clinically, tumour it is good it is pernicious be an important factor for judging dog tumor of breast prognosis.And detect tumour mark
Will object can help to judge the property of tumour and prognosis situation, but pet clinic is more rare at present.Infiltration, the transfer of tumour relate to
And to multifactor and multiple the step of recurring, there is the different factors to participate in each stage, for example, cell adhesion because
Son, matrix metalloproteinase etc..
The tumor markers clinically used at present have many flaws: 1. specificity and sensitivity are not high enough, exist
Mistaken diagnosis or the case where fail to pinpoint a disease in diagnosis；2. testing cost is higher, Combining diagnosis is difficult to popularize in common physical examination；3. certain tumours are not yet
The marker of specificity.
Summary of the invention
The purpose of the invention is to overcome the deficiencies of the prior art and provide one kind to can be used for dog Diagnosis of Breast Tumor and pre-
The molecular marker judged afterwards.
The first purpose of the invention is to provide a kind of molecular markers that can be used for dog Diagnosis of Breast Tumor and Index for diagnosis
Object.
Molecule mark a second object of the present invention is to provide ICAM1 gene as dog Diagnosis of Breast Tumor and Index for diagnosis
The application of will object.
Third object of the present invention is to provide the RT-qPCR detection primers of a pair of of detection ICAM1 gene expression.
Fourth object of the present invention is to provide the RT-qPCR detection primer in preparation dog Diagnosis of Breast Tumor and prognosis
Judge the application in kit.
Fifth object of the present invention is to provide the kits of a kind of dog Diagnosis of Breast Tumor and Index for diagnosis.
To achieve the goals above, the present invention is achieved by the following technical programs:
A kind of molecular marker can be used for dog Diagnosis of Breast Tumor and Index for diagnosis, the molecular marker are ICAM1
Gene, nucleotide sequence is as shown in SEQ ID NO:1.
Application of the ICAM1 gene as dog Diagnosis of Breast Tumor and the molecular marker of Index for diagnosis.
The RT-qPCR detection primer of a pair of detection ICAM1 gene expression, the nucleotide sequence such as institute of SEQ ID NO:2~3
Show.
Application of the RT-qPCR detection primer described above in preparation dog Diagnosis of Breast Tumor and Index for diagnosis kit,
Also belong to protection scope of the present invention.
A kind of kit of dog Diagnosis of Breast Tumor and Index for diagnosis, the kit, which contains, is able to detect ICAM1 gene
Expression quantity reagent.
Preferably, the reagent of the expression quantity for being able to detect ICAM1 gene is the above RT-qPCR primer.
Preferably, the kit also contains ddH2O, the detection primer of SYBR Green I Master and reference gene.
Preferably, the reference gene is β-actin, the nucleotide sequence of RT-qPCR detection primer such as SEQ ID
Shown in NO:4~5.
Most preferably, a kind of kit of dog Diagnosis of Breast Tumor and Index for diagnosis contains detection ICAM1 gene expression
RT-qPCR detection primer, nucleotide sequence is as shown in NO:2~3 SEQ ID；The RT-qPCR that reference gene is β-actin is examined
Primer is surveyed, the nucleotide sequence of RT-qPCR detection primer is as shown in NO:4~5 SEQ ID；Also contain ddH2O、SYBR
GreenⅠMaster。
The 20 μ L of RT-qPCR system total volume of kit described above, specifically: ddH2O 7.2μL；SYBR GreenⅠ
Master 10μL；0.4 μ L of forward primer；It was found that 0.4 μ L of primer；2 μ L of DNA profiling.
The RT-qPCR response procedures of kit described above are as follows:
Kit application method described above the following steps are included:
S1. sample total serum IgE is extracted；
S2. cDNA is synthesized；
S3. RT-qPCR is carried out.
Compared with prior art, the invention has the following beneficial effects:
Only significant low expression (P < 0.05), this low expression do not go out ICAM1 gene in benign tumour in malignant tumour
Existing significant difference (P > 0.05) illustrates that ICAM1 can be used as the molecular labeling for identifying dog Breast Tumors.It is further bright
Effect of the true ICAM1 gene in the transfer of dog tumor of breast, carries out RT-qPCR to dog breast tumor cell line CHMp and CHMm,
ICAM1 gene expression amount in the CHMm cell of transfer stove significant difference (P < 0.05) compared with CHMp cell as the result is shown, into
One step illustrates that ICAM1 gene takes part in the infiltration of dog tumor of breast, transfer process, and it is good that ICAM1 can be used as identification dog tumor of breast
Pernicious molecular labeling.Therefore, ICAM1 can be used as the molecular marker of dog Diagnosis of Breast Tumor and Index for diagnosis.The present invention
The molecular marker relevant to the transfer of dog tumor of breast provided, sensibility and specificity with higher, to dog tumor of breast
Property identification, prognosis evaluation, in time determine therapeutic scheme, improve dog only survival rate have great importance.
Detailed description of the invention
Fig. 1 is β-actin gene melting curve.
Fig. 2 is ICAM1 gene melting curve.
Fig. 3 is malignant tumors group ICAM1 gene expression amount difference；Ordinate indicates cancerous tissue compared to corresponding cancer beside organism
The variation multiple of ICAM1 expression；Control Zu Zhiquan breast cancer cancer beside organism.
Fig. 4 is benign tumour group ICAM1 gene expression amount difference；Ordinate indicates neoplastic lesion tissue compared to corresponding disease
Become the variation multiple of surrounding tissue ICAM1 gene expression dose；Control group refers to dog tumor of breast lesion surrounding tissue.
Fig. 5 is ICAM1 gene expression amount difference in CHMm and CHMp cell；Ordinate indicate CHMm cell compared to
The variation multiple of ICAM1 gene expression dose in CHMp cell；Control group refers to CHMp cell.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Selected sample of breast tissue is all from each pets hospital in Guangzhou and receives mastectomy in following embodiment
Suffer from dog, all sample standard deviations do not receive radiation and chemotherapy and exclude other tumor types.
1, experimental material
CHMp cell line is that separation is established from the primary tumor for the female experimental dog for suffering from tumor of breast, CHMm cell line
It is that separation is established from the transfer stove for the female experimental dog for suffering from tumor of breast.This two plants of cell lines are by Tokyo Univ Japan's agriculture
The present of industry portion life science subject dog institute's structive sursery laboratory, is now stored in this laboratory.
2, the Primary Chemical and biological agent details used is shown in Table 1.
1 Primary Chemical of table and biological agent:
3, the major experimental instrument used
Details are shown in Table 2.
2 major experimental instrument of table:
4, the statistics and analysis of test data
Each experimental group data are all made of the analysis of Excel 2010.Each group mean is the average value tested three times, two groups
Compare between mean and examined using t, test stone is to have significant difference, the generation of P≤0.01 between P≤0.05 represents each group of data
There is extremely significant sex differernce between table each group of data." * " represents 0.01 < P≤0.05 in each figure；" * * " represents P≤0.01.Mapping
Using 6 software of GraphPad Prism.
1 RT-qPCR method of embodiment detects mRNA expression of the ICAM1 in dog breast tumor tissues
One, experimental method
1, sample storage and processing
Cancer group is separated after the fresh tumor tissue of operation excision is rinsed well tissue surface bloodstain and hair etc. with PBS
Knit and cancer beside organism (apart from cancerous swelling lesion≤2cm) and be broken down into 1cm × 1cm tissue block be placed in sterilizing EP pipe after immediately
With liquid nitrogen frozen, it is subsequently placed at -80 DEG C and saves backup；Each sample takes portion of tissue to fix use with the paraformaldehyde of 4% concentration
In it is subsequent do specimens paraffin embedding slices and send Germany receive Boke face animal clinical examine laboratory qualification.
2, the production of pathological section and HE dyeing
Consolidate after dog breast tumor tissues sample progress specimens paraffin embedding slices and HE that clinic is collected are dyed with paraformaldehyde
The tissue block set send together Germany receive Boke face animal clinical examine laboratory identified.
Specimens paraffin embedding slices
(1) it draws materials: being taken out secure for 24 hours above tissue by 4% paraformaldehyde, with scalpel by required position
Stripping and slicing (thickness is no more than 0.5cm) is simultaneously placed in dewatering box with corresponding label together after tissue equating is whole.
(2) it is dehydrated: 75% alcohol of alcohol 4h → 85% alcohol of 2h → 90% alcohol of 2h → 95% 1h → dehydrated alcohol I
30min → II 30min of dehydrated alcohol → alcohol benzene 5min~10min → I 5min of dimethylbenzene~10 min → dimethylbenzene, II 5min~
10min → I 1h of wax → II 1h of wax → wax, III 1h.
(3) it embeds: the wax melted is put into embedding frame, tissue is put into embedding frame before wax solidification and is sticked
Corresponding label takes out wax stone after being placed in -20 DEG C of jelly platforms coolings and is modified.
(4) it is sliced: paraffin mass is cut into the slice with a thickness of 4 μm.
(5) it bakes piece: the piece cut being picked up after flattening on 40 DEG C of warm water, 60 DEG C of drying in oven is put into, is finally placed in
It is stored at room temperature spare.
HE Coloration experiment step
(1) paraffin section de-waxing is to water: I 20min of dimethylbenzene → II 20min of dimethylbenzene → I 10min of dehydrated alcohol → nothing
The water-ethanol alcohol of II 10min → 95% alcohol of 5min → 90% alcohol of 5min → 80% alcohol of 5min → 70% 5min → distillation
Washing.
(2) bush uniformly dyeing nucleus: Harris bush uniformly dyeing 3min~8min → flowing water rinses → 1% hydrochloride alcohol point
Change several seconds → flowing water → 0.6% ammonium hydroxide of flushing to return indigo plant → flowing water and rinse.
(3) Yihong contaminates cytoplasm: eosin stain contaminates 1min~3min.
(4) it is dehydrated mounting: 95% II 5min of the alcohol I alcohol of 5min → 95% → I 5min of dehydrated alcohol → dehydrated alcohol
II 5min → I 5min of dimethylbenzene → II 5min of dimethylbenzene → slice slightly dries → neutral gum mounting.
3, mRNA expression of the RT-qPCR method detection ICAM1 in dog breast tumor tissues
(1) tissue sample pre-processes
Test serum sample 50mg~100mg is taken to be put into the homogenate tube crossed through high pressure sterilization, 1 ml is added in every pipe.
(2) extraction of total tissue RNA
Execution is strictly operated according to Guangzhou Dongsheng Biotechnology Co., Ltd total RNA extraction reagent box specification.
(3) synthesis of cDNA
The reverse transcription system (table 3) of total serum IgE is prepared referring to Takara reverse transcription reagent box.
3 reverse transcription system of table:
(4)RT-qPCR
The cDNA of reverse transcription is taken out, according to RocheThe requirement of real-time PCR establishes 20
The system (table 4) of μ L carries out the RT-qPCR detection of ICAM1 (its nucleotide sequence is as shown in SEQ ID NO:1), β-actin
For reference gene.Fluorescence quantification PCR primer sequence is (table 5) as shown in the table:
The system of 4 fluorescent quantitation of table
5 RT-qPCR primer sequence of table
It is protected from light according to required PCR hole count and prepares PCR mix, each hole is separately added into after mixing well, to reduce loading errors,
Each detection gene of each sample sets three repetitions.
After sample-adding, with sealing plate film sealing plate, plate is put by 1500 × g after being centrifuged 96 orifice plate 2min of PCRIn instrument, according to following parameter setting:
RT-qPCR interpretation of result:
The experimental result calculated using following formula:
2-ΔΔct
In formula-Δ Δ ct=- [(experimental group target gene ct value-experimental group reference gene ct value)-(control group purpose
Gene ct value-control group reference gene ct value)]
Two, experimental result
The dog Breast Tumor Samples that clinic is collected are divided into malignant tumors group and benign by Histopathology testing result
Tumor group；Take the tissue of lesion locations with corresponding perilesional tissue and using the method for RT-qPCR detection ICAM1's respectively
MRNA expression, β-actin are reference gene.The melting curve of β-actin gene and ICAM1 gene such as Fig. 1 and Fig. 2 institute
Show.
Malignant tumors group RT-qPCR is as the result is shown: ICAM1 is by the mrna expression amount in dog breast cancer tissue is relative to cancer
Tissue is lowered, and significant difference (P < 0.05), such as Fig. 3.
Benign tumour group RT-qPCR is as the result is shown: mrna expression amount split-phase pair of the ICAM1 in dog tumor of breast lesion tissue
It is lowered in perilesional tissue, but there were significant differences (P > 0.05), such as Fig. 4.
ICAM1 gene only occurs in malignant tumour significant low expression (P < 0.05), this low expression benign tumour not
Occur significant difference (P > 0.05), prompts ICAM1 may be as the potential molecular labeling for identifying dog Breast Tumors.
2 RT-qPCR method of embodiment detects mRNA expression of the ICAM1 in CHMm and CHMp cell
One, the culture of dog breast tumor cell CHMm and CHMp
1, the recovery of dog breast tumor cell CHMm and CHMp
Being put into rapidly after CHMp and CHMm cell is removed from liquid nitrogen in 37 DEG C of water-baths and constantly shaking makes it quickly
Dissolution；It is transferred them in 15mL centrifuge tube after cell in cryopreservation tube all melts, every pipe is added 10mLDMEM and blows repeatedly
It beats and mixes；Subsequent 2,000rpm is centrifuged 5min；Abandon supernatant, with the complete cell culture medium of 1mL (90%DMEM, 10% serum, 1%
It is dual anti-) it suspension cell and transfers them in 25cm2 attached cell culture bottle, every bottle of addition 4mL complete medium postposition again
It is cultivated in 37 DEG C, the incubator containing 5%CO2.It is observed under the microscope after for 24 hours, if most cells are normal adherent, only
There is small part cells float in cell bottle, illustrates cell recovery success.
2, the passage of dog breast tumor cell
CHMp and CHMm cell is adherent growth cell, when observing that cell density reaches about 90% under the microscope,
Then passed on.Original culture medium in Tissue Culture Flask is discarded first, uses liquid relief after 2mL~3mL PBS buffer solution is added
Device discards after gently purging the adherent surface of cell, washs 1~2 time.Then the trypsase containing 0.25%EDTA is added to carry out
Digestion, laying flat cell bottle and shaking gently makes trypsase uniform fold attached cell face；It is observed when under inverted microscope
When cellular morphology is rounded, space between cells increases, trypsase is sucked out.Appropriate training completely is added into cell bottle according to cell density
It supports base and simultaneously blows and beats bottle wall cell, need for cell suspension to be dispensed into according to experiment and carry out passage in new attached cell bottle and will be thin
Born of the same parents' bottle is placed in 37 DEG C, cultivates in the cell incubator of 5%CO2；About 1d~2d passage is primary.
3, dog breast tumor cell freezes
The cell cryopreservation box equipped with sufficient isopropanol are placed at room temperature for after taking out in -80 DEG C of refrigerators in advance makes its heating；
The cell covered with (density reaches 90% or more) is taken out from incubator, PBS is added after washing 1~2 time to be contained
The trypsin digestion and cell of 0.25%EDTA；Pancreatin is discarded, blown and beaten the cell in adherent face with DMEM and is transferred to 15mL
In centrifuge tube, 2,000rpm centrifugation 5min；Supernatant is abandoned, it is anti-with the configured cells frozen storing liquid of 1 mL (10%DMSO, 90%FBS)
Multiple piping and druming makes cell suspend again and is transferred in cryopreservation tube, marks and seals with sealing film；Cryopreservation tube is put into gradient to freeze
It deposits in box and is immediately placed in -80 DEG C of refrigerators and save.Cell can be transferred in liquid nitrogen container and be saved after for 24 hours.
Two, mRNA expression of the RT-qPCR method detection ICAM1 in CHMp and CHMm cell
1, sample pretreatment
CHMp and CHMm cell is uniformly laid in 6 orifice plates, every kind of cell sets three repetitions.Cell is collected after 24 h of bed board
Carry out next step experiment.
2, the extraction of cell total rna
(1) cell sample pre-processes
Old culture medium is discarded, is directly added in 6 orifice plates according to every hole 1mL after washing 1~2 time with PBS buffer solution molten
Liquid RL lytic cell is simultaneously repeatedly blown and beaten with pipettor.
(2) extraction of cell total rna
With embodiment 1.
(3) synthesis of cDNA
With embodiment 1.
(3)RT-qPCR
With embodiment 1.
Two, experimental result
Dog breast tumor cell line CHMp is to separate foundation from the female experimental dog for suffering from tumor of breast with CHMm,
CHMp is primary lesion cell, and CHMm is metastatic lesion cell.Using the method detection ICAM1 of RT-qPCR in CHMm cell and
MRNA expression difference in CHMp cell, wherein CHMp cell is control group.ICAM1 is in CHMm cell as the result is shown
Mrna expression amount lowered compared to CHMp cell, and significant difference (P < 0.05), such as Fig. 5.
The kit of embodiment 3 a kind of dog Diagnosis of Breast Tumor and Index for diagnosis
One, a kind of kit of dog Diagnosis of Breast Tumor and Index for diagnosis
A kind of kit of dog Diagnosis of Breast Tumor and Index for diagnosis, the RT-qPCR containing detection ICAM1 gene expression
Detection primer, nucleotide sequence is as shown in NO:2~3 SEQ ID；The RT-qPCR detection that reference gene is β-actin is drawn
Object, the nucleotide sequence of RT-qPCR detection primer is as shown in NO:4~5 SEQ ID；Also contain ddH2O、SYBR GreenⅠ
Master。
Two, the application method of kit
Kit application method are as follows: extract sample total serum IgE, synthesize cDNA, carry out RT-qPCR.
S1. sample total serum IgE is extracted；
S2. cDNA is synthesized；
S3. RT-qPCR is carried out.
Wherein, 20 μ L of RT-qPCR system total volume, specifically: ddH2O 7.2μL；SYBR GreenⅠMaster 10μL；
0.4 μ L of forward primer；It was found that 0.4 μ L of primer；2 μ L of DNA profiling.
Wherein, RT-qPCR response procedures are as follows:
。
Sequence table
<110>Agricultural University Of South China
<120>a kind of molecular marker that can be used for dog Diagnosis of Breast Tumor and Index for diagnosis
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1596
<212> DNA
<213> Canis lupus familiaris
<400> 1
atggctcccg gccccgccgc gcccgcgctg cccgcgctcc tggccctcct cggggctctg 60
ctcccaggac ttggaggtgc ccagacgtcc gtggacccag cagaagccat cataccccga 120
ggaggctctg tgcaggtgaa ctgtagtacc tcatgcaacc agacctccat cttcggcctg 180
gagactctgt tgactaagaa ggaactgaac tctggggaca actgggtgct ctttgaactg 240
actgatgtgc aagaagatag caagctgata tgtttctcaa actgtcatga gcagaccatg 300
gctccgatgc acctcaccgt atactggttc ccggagcgag tggagctggc acccctaccc 360
cgctggcagc ccgtgggtga gaacctcacc atgacctgcc aggtggcagg cggggcgccc 420
cggaccaacc tcacggtggt gctgctccgc ggggaggagg agctgagccg gcaaccggcc 480
gtcggggagc cagccgaggt cacgttcacg gtggcggtgg gcagagagga ccacctcgcc 540
aacttctcgt gccgcacgga cctggacctg aggcaccgag ggttgggatt gttccagaac 600
agctcggcgc ccaggcagct ccaaaccttt gtcctgccag agaccccccc acgccttgct 660
acccccccga ttgtggaagt gggcacacag tggtctgtgg actgcactct ggatggggtg 720
ttcccagctt cggaggccca agtccacctg gcgttagcag aagagaggct gcactccaca 780
gtcctgtaca aaaaggactc cctcttggcc acggcaaatg tcaaagcaaa cccagaagac 840
gagggtaccc agcagctatg gtgtgaggtg acactgggag acgagaatcg gaggtggcag 900
gagaatgtga ccttctatag cttcccggca cccaacctga ccctgagtga gccagaggtc 960
tcagaatgga ctacggtgac tgtggagtgt gaggcccctg ctggagtcgt ggtgtcgctg 1020
agcgggctcc cttcggggct tgcagtaccc agggcacagt tccaactaaa tgccagcgct 1080
gccgacaaca ggcgcagctt ctcctgctct gctgccctgg aggtggctgg gcacatgctg 1140
caaaagaacc agacccggga gctccacgtc ctgtatggtc cccgactaga ccagagggat 1200
tgtccgggaa actggacgtg ggaggaaggc tcccatcaga ccctgaagtg ccaagcttgg 1260
gggaacccgg ttcctgagct gaaatgtcac cggaaggggg atgatgcttt gctccccatc 1320
ggggacctga gacctgtcaa gcgggaggtt gctggcactt acctgtgtca ggcccggagc 1380
ccccgtggtg agatcacccg agaagtggtc atcaacgtga tctaccacca gaacaacatt 1440
cttatcatca tcctggtgac aaccattgtc atcttaggaa ctgtgagtgt agccgcttac 1500
ctctataacc gccagcggaa gatccagaaa tacaagcttc agaaggccca ggaggcagct 1560
gccatgaagc tgaacacacc ggccacaccc ccctga 1596
<210> 2
<211> 20
<212> DNA
<213> Canis lupus familiaris
<400> 2
ccaccccctg aagctaagtc 20
<210> 3
<211> 20
<212> DNA
<213> Canis lupus familiaris
<400> 3
cttgcggatg tcaacgtcac 20
<210> 4
<211> 22
<212> DNA
<213> Canis lupus familiaris
<400> 4
tcggaggtgg caggagaatg tg 22
<210> 5
<211> 20
<212> DNA
<213> Canis lupus familiaris
<400> 5
cagcgacacc acgactccag 20
Claims (8)
1. a kind of molecular marker that can be used for dog Diagnosis of Breast Tumor and Index for diagnosis, which is characterized in that the molecular marker
Object is ICAM1 gene, and nucleotide sequence is as shown in SEQ ID NO:1.
2. application of the ICAM1 gene as dog Diagnosis of Breast Tumor and the molecular marker of Index for diagnosis.
3. the RT-qPCR detection primer of a pair of detection ICAM1 gene expression, which is characterized in that its nucleotide sequence such as SEQ ID
Shown in NO:2~3.
4. the answering in preparation dog Diagnosis of Breast Tumor and Index for diagnosis kit of RT-qPCR detection primer described in claim 3
With.
5. a kind of kit of dog Diagnosis of Breast Tumor and Index for diagnosis, which is characterized in that the kit, which contains, to be able to detect
The reagent of the expression quantity of ICAM1 gene.
6. kit according to claim 6, which is characterized in that the reagent of the expression quantity for being able to detect ICAM1 gene
For RT-qPCR primer described in claim 3.
7. kit according to claim 7, which is characterized in that the kit also contains ddH2O、SYBR Green Ⅰ
The detection primer of Master and reference gene.
8. kit according to claim 7, which is characterized in that the reference gene is β-actin, RT-qPCR detection
The nucleotide sequence of primer is as shown in NO:4~5 SEQ ID.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810737827.9A CN109022575A (en) | 2018-07-06 | 2018-07-06 | A kind of molecular marker can be used for dog Diagnosis of Breast Tumor and Index for diagnosis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810737827.9A CN109022575A (en) | 2018-07-06 | 2018-07-06 | A kind of molecular marker can be used for dog Diagnosis of Breast Tumor and Index for diagnosis |
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Family
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116083590A (en) * | 2023-03-23 | 2023-05-09 | 广州爱仁生物医药科技有限公司 | Gene detection kit and preparation method and application thereof |
-
2018
- 2018-07-06 CN CN201810737827.9A patent/CN109022575A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| S M SHEEN-CHEN等: "Serum levels of circulating intercellular adhesion molecule-1 in patients with breast cancer", 《ANTICANCER RES》 * |
| 何俊玲: "ICAM-1在人乳腺良恶性肿瘤组织中的表达研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 未知: "PREDICTED: Canis familiaris intercellular adhesion molecule-1 (ICAM-1), mRNA", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116083590A (en) * | 2023-03-23 | 2023-05-09 | 广州爱仁生物医药科技有限公司 | Gene detection kit and preparation method and application thereof |
| CN116083590B (en) * | 2023-03-23 | 2024-03-22 | 雄安妙心医学检验有限公司 | Gene detection kit and preparation method and application thereof |
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