CN107058523A - A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application - Google Patents
A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application Download PDFInfo
- Publication number
- CN107058523A CN107058523A CN201710168670.8A CN201710168670A CN107058523A CN 107058523 A CN107058523 A CN 107058523A CN 201710168670 A CN201710168670 A CN 201710168670A CN 107058523 A CN107058523 A CN 107058523A
- Authority
- CN
- China
- Prior art keywords
- breast carcinoma
- risk assessment
- seq
- genes
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- 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
Abstract
The present invention relates to a kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application, including SEQ ID NO.：Nucleotide sequence shown in 1~42, belongs to field of molecular detection.The present invention carries out augmentation detection, so as to consider whether to need the clinical measures such as NACT using fluorescent quantitative poly chain reaction using the advantage of fluorescent quantitation to 21 genes of breast carcinoma recurring risk assessment.The kit provides the pcr amplification primer thing sequence of 21 genes of breast carcinoma recurring risk assessment, it is adaptable to which ER is positive, HER2 is negative, axillary gland is negative, tumor size is for low differentiation in 0.6~1.0cm or with poor prognosis factor person, or tumour>The detection of 1cm person, have the advantages that sensitivity and specificity it is high, stably, it is timely, easy to operate, can preferably meet the clinical practice of breast carcinoma recurring risk assessment, can reduce invalid medication, raising medication accuracy rate, reduction patient economy burden.
Description
Technical field
The invention belongs to field of molecular detection, and in particular to one kind is exclusively used in the gene of breast carcinoma recurring risk assessment 21
Nucleotide sequence detection kit.
Background technology
Breast cancer is the major malignant tumor for endangering WomanHealth, and the whole world there are about 1,200,000 women every year and suffer from breast cancer, 50
Ten thousand people die from breast cancer.It is 2.61/10 ten thousand in the average mortality of China's breast cancer, it is notable with the death rate in coastal several cities
Higher, the death rate in city is higher by 1.4 times than rural area.Its incidence of disease was in ascendant trend year by year in recent years for China, although clinical
Research has been achieved for larger progress, but its recurrence and metastasis rate is still very high.Over nearly 20 years, centered on operation, change
Treatment, radiotherapy, endocrine therapy obtain significant progress for the multidisciplinary synthesis Therapeutic mode of auxiliary, make the recurrence of breast cancer and dead
The rate of dying is significantly reduced, but still the effective method of shortage can relatively accurately predict the risk of recurrence of patient and give corresponding
Treatment.
21 gene recurring risk assessments are a kind of according to 21 gene associations detections, pass through certain formula and calculate recurrence fraction
Method to judge breast carcinoma recurring risk.It is made up of 16 cancer related genes and 5 reference genes.Cancer related gene
It is broadly divided into 5 classes, respectively proliferation-associated genes (Ki67, STK15, Survivin, CCNB1 and MYBL2), EGF
Acceptor related gene (GRB7 and HER2), hormone related gene (ER, PgR, BCL2 and SCUBE2), infiltration related gene (MMP11
And CTSL2) and 3 unfiled genes (GSTM1, CD68 and BAG1).5 reference genes be respectively ACTB, GAPDH, RPLPO,
GUS and TFRC.The selection of said gene is according to cancer pertinent literature, microarray data data, genomic database and divided
Sub- biological information selectes 250 genes and is used as candidate gene.According to the research of 3 independent clinical tests totally 447 patients
As a result, choose with recurring significantly correlated gene at a distance, 250 candidate genes are further screened, above-mentioned 16 are finally determined
Individual cancer related gene, and 5 house-keeping genes are determined as reference gene.This detection suitable for Lymph Node-negative, it is female swash
Plain receptor positive and in the future may receive clinical I, the II primary breast cancer patient of tamoxifen treatment.Pass through 21 genes of analysis
Express to determine RS score values (0~100 point), RS<18th, 18≤RS≤30, RS >=31 are respectively basic, normal, high danger patient.2008
Year,《US National integrates cancer network (NCCN) breast cancer clinical practice guideline》Point out, for ER is positive, HER2 is negative, armpit
Nest Lymph Node-negative, tumor size are for low differentiation in 0.6~1.0cm or with poor prognosis factor person, or tumour>1cm person, examines
Consider and patient's row is further analyzed using RS, give high-risk person adjuvant chemotherapy of patients (2B classes).
Real-Time Fluorescent Quantitative PCR Technique (Real-time quantitative Polymerase Chain Reaction
Abbreviation Real Time PCR) it is the nucleic acid quantitation technique grown up in qualitative PCR technical foundation, in PCR reaction systems
Fluorophor is added, whole PCR processes are monitored in real time using fluorescence signal accumulation, finally by Ct values and standard curve to sample
In the methods that are quantitatively detected of DNA (or cDNA).The technology is not only realized to be quantified to DNA/RNA templates, and tool
Have that sensitivity and specificity are high, the features such as to realize multiple reaction, automaticity high, pollution-free, with low cost.It is existing at present
The kit of a variety of utilization quantitative PCR techniques is applied to the related pathogen detection of clinical disease, Genotyping, mutation research
Deng.
The present invention using fluorescent quantitation advantage, with easy to operate, high specificity, sensitiveness is high, with low cost and height
The advantages of flux, 21 genes of breast carcinoma recurring risk assessment are expanded simultaneously using fluorescent quantitative poly chain reaction
Detection, so as to consider whether to need the clinical treatment measure such as NACT.
The content of the invention
The purpose of the present invention is a kind of sensitive, accurate, easy to operate new detection kit of exploitation, realizes that breast cancer is answered
21 Gene Detectings of risk assessment are sent out, to make up the deficiency of existing detection method.
A kind of genetic test primer of breast carcinoma recurring risk assessment 21, including SEQ ID NO.：Nucleotides shown in 1~42
Sequence.
Specific primer amplification information is as follows：
(1) specific primer of cancer related gene Ki67 genes, such as SEQ ID NO are expanded：1 and SEQ ID NO：2 institutes
Show；
(2) specific primer of cancer related gene STK15 genes, such as SEQ ID NO are expanded：3 and SEQ ID NO：4 institutes
Show；
(3) specific primer of cancer related gene Survivin genes, such as SEQ ID NO are expanded：5 and SEQ ID NO：
Shown in 6；
(4) specific primer of cancer related gene CCNB1 genes, such as SEQ ID NO are expanded：7 and SEQ ID NO：8 institutes
Show；
(5) specific primer of cancer related gene MYBL2 genes, such as SEQ ID NO are expanded：9 and SEQ ID NO：10
It is shown；
(6) specific primer of cancer related gene GRB7 genes, such as SEQ ID NO are expanded：11 and SEQ ID NO：12
It is shown；
(7) specific primer of cancer related gene HER2 genes, such as SEQ ID NO are expanded：13 and SEQ ID NO：14
It is shown；
(8) specific primer of cancer related gene ER genes, such as SEQ ID NO are expanded：15 and SEQ ID NO：16 institutes
Show；
(9) specific primer of cancer related gene PgR genes, such as SEQ ID NO are expanded：17 and SEQ ID NO：18 institutes
Show；
(10) specific primer of cancer related gene BCL2 genes, such as SEQ ID NO are expanded：19 and SEQ ID NO：20
It is shown；
(11) specific primer of cancer related gene SCUBE2 genes, such as SEQ ID NO are expanded：21 and SEQ ID NO：
Primer pair shown in 22；
(12) specific primer of cancer related gene MMP11 genes, such as SEQ ID NO are expanded：23 and SEQ ID NO：
Shown in 24；
(13) specific primer of cancer related gene CTSL2 genes, such as SEQ ID NO are expanded：25 and SEQ ID NO：
Primer pair shown in 26；
(14) specific primer of cancer related gene GSTM1 genes, such as SEQ ID NO are expanded：27 and SEQ ID NO：
Primer pair shown in 28；
(15) specific primer of cancer related gene CD68 genes, such as SEQ ID NO are expanded：29 and SEQ ID NO：30
It is shown；
(16) specific primer of cancer related gene BAG1 genes, such as SEQ ID NO are expanded：31 and SEQ ID NO：32
It is shown；
(17) specific primer of reference gene ACTB genes, such as SEQ ID NO are expanded：33 and SEQ ID NO：34 institutes
Show；
(18) specific primer of reference gene GAPDH genes, such as SEQ ID NO are expanded：35 and SEQ ID NO：36 institutes
Show；
(19) specific primer of reference gene RPLPO genes, such as SEQ ID NO are expanded：37 and SEQ ID NO：38 institutes
Show；
(20) specific primer of reference gene gus gene, such as SEQ ID NO are expanded：39 and SEQ ID NO：Shown in 40；
(21) specific primer of reference gene TFRC genes, such as SEQ ID NO are expanded：41 and SEQ ID NO：42 institutes
Show.
A kind of gene detecting kit of breast carcinoma recurring risk assessment 21, the kit includes SEQ ID NO.：1~42
Shown primer.
The above-mentioned genetic test primer of breast carcinoma recurring risk assessment 21 applying at this in breast carcinoma recurring risk assessment
Within the protection domain of invention.
A kind of gene tester of breast carcinoma recurring risk assessment 21, comprises the following steps：
(1) paraffin organization RNA is extracted；
(2) reverse transcription；
(3) fluorescent quantitative PCR；
(4) interpretation of result：Judge that recurrence prediction risk is specific according to following standard：Low-risk：RS≤18；Risk：18
＜ RS ＜ 31；Excessive risk：RS≥31.
In step (2), reverse transcription reaction system is 20 μ L, wherein, 11 μ L, RT enzyme of template ribonucleic acid, 1 μ L, RI enzyme 1 μ L, dNTP
2 μ L, 5 × buffer 4 μ L, the μ L of random primer 1；
Reverse transcription reaction condition：42 DEG C of 30min, 95 DEG C of 5min.
In step (3), fluorescent quantitative PCR system is：PCR reaction systems are 20 μ L, wherein the μ L of 2 × Mix 10, on
Trip and mixed downstream primer (10 μm of ol/L) 0.4 μ L, the μ L of template ribonucleic acid 2, pyrocarbonic acid diethyl ester (DEPC) processing water 7.6 μ L；
Quantitative fluorescent PCR reaction condition is：94 DEG C of 20s, 60 DEG C of 15s, extend 72 degree of 15s, fluorescent collecting o'clock is at 60 DEG C, instead
Volume is answered to be set to 20 μ L.
The nucleotide sequence is by 21 pairs of primer sets to 16 cancer related genes and 5 reference genes progress augmentation detections
Into.The cancer related gene is broadly divided into 5 classes, respectively proliferation-associated genes (Ki67, STK15, Survivin, CCNB1 and
MYBL2), EGF-R ELISA related gene (GRB7 and HER2), hormone related gene (ER, PgR, BCL2 and
SCUBE2), infiltration related gene (MMP11 and CTSL2) and 3 unfiled genes (GSTM1, CD68 and BAG1).5 ginsengs
It is respectively ACTB, GAPDH, RPLPO, GUS and TFRC to examine gene.
Beneficial effect：
(1) present invention using fluorescent quantitation advantage, with easy to operate, high specificity, sensitiveness it is high, with low cost and
The advantages of high flux.
(2) 21 pairs of primer output fragments involved in the present invention substantially increase paraffin specimen extraction all within 150bp
PCR efficiency after mRNA reverse transcriptions.
(3) 21 pairs of primers involved in the present invention its amplification condition when entering performing PCR is consistent, will substantially reduce detection real
Test the time.
Amplification inspection is carried out to 21 genes of breast carcinoma recurring risk assessment using fluorescent quantitative poly chain reaction simultaneously
Survey, so as to consider whether to need the clinical treatment measure such as NACT.The kit and technical method have easy to operate, special
The strong, sensitiveness of property is high, with low cost and the advantages of high flux, quick detection breast carcinoma recurring risk can be realized by fluorescent quantitation
21 genes assessed, reference is provided for clinical treatment.
Brief description of the drawings
Fig. 1 is 21 genetic test electrophoretograms of 2 breast carcinoma recurring risk assessments, can be amplified under identical conditions,
And the formation without primer dimer.
Fig. 2 is the quantitative fluorescent PCR figure of 21 genetic tests of 1 breast carcinoma recurring risk assessment, its CT value 25-40 it
Between, abscissa is period in figure；Ordinate is fluorescent value.
Embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply example and be merely to illustrate the present invention, without should be also without limitation on the present invention described in detail in claims.
Embodiment 1：Detect the nucleotide sequence of the gene of 21 genetic tests of breast carcinoma recurring risk assessment.
21 pairs of specific primer commission Invitrogen (Shanghai) Trading Co., Ltd. synthesis：
SEQ ID NO：1 5′-CGGACTTTGGGTGCGACTT-3′
SEQ ID NO：2 5′-TTACAACTCTTCCACTGGGACGAT-3′
SEQ ID NO：3 5′-GCTAGAGGCATCATGGACCGA-3′
SEQ ID NO：4 5′-GAAGGACACAAGACCCGCTGA-3′
SEQ ID NO：5 5′-AGAACTGGCCCTTCTTGGAGG-3′
SEQ ID NO：6 5′-TCTATGGGGTCGTCATCTGGC-3′
SEQ ID NO：7 5′-TTCAGGTTGTTGCAGGAGAC-3′
SEQ ID NO：8 5′-CATCTTCTTGGGCACACAAT-3′
SEQ ID NO：9 5′-GCCGAGATCGCCAAGATG-3′
SEQ ID NO：10 5′-CTTTTGATGGTAGAGTTCCAGTGATTC-3′
SEQ ID NO：11 5′-CCATCTGCATCCATCTTGTT-3′
SEQ ID NO：12 5′-GGCCACCAGGGTATTATCTG-3′
SEQ ID NO：13 5′-CGGTGTGAGAAGTGCAGCAA-3′
SEQ ID NO：14 5′-CCTCTCGCAAGTGCTCCAT-3′
SEQ ID NO：15 5′-TGTCCAGCCACCAACCAGTG-3′
SEQ ID NO：16 5′-CCCTCCTCTTCGGTCTTTTCG-3′
SEQ ID NO：17 5′-GCATCAGGCTGTCATTATGG-3′
SEQ ID NO：18 5′-AGTAGTTGTGCTGCCCTTCC-3′
SEQ ID NO：19 5′-ATCGCCCTGTGGATGACTGAG-3′
SEQ ID NO：20 5′-ATGCTGGGGCCGTACAGTTC-3′
SEQ ID NO：21 5′-TGACAATCAGCACACCTGCAT-3′
SEQ ID NO：22 5′-TGTGACTACAGCCGTGATCCTTA-3′
SEQ ID NO：23 5′-CCCAGGGCCACATTTGGTT-3′
SEQ ID NO：24 5′-GGACTGGCTTTTCACCGTCGT-3′
SEQ ID NO：25 5′-TGAAAGCAGTCGCAACTGTGG-3′
SEQ ID NO：26 5′-CGAATTTGCTCCTTCAAAGCC-3′
SEQ ID NO：27 5′-GGGACGCTCCTGATTATGAC-3′
SEQ ID NO：28 5′-GTGAGCCCCATCAATCAAGT-3′
SEQ ID NO：29 5′-GCAGCACAGTGGACATTCTCG-3′
SEQ ID NO：30 5′-AACTGAAGCTCTGCCCCAGG-3′
SEQ ID NO：31 5′-CGTTGTCAGCACTTGGAATACAA-3′
SEQ ID NO：32 5′-GTTCAACCTCTTCCTGTGGACTGT-3′
SEQ ID NO：33 5′-TTCCTTCCTGGGCATGGAGTC-3′
SEQ ID NO：34 5′-GGTCTTTGCGGATGTCCACG-3′
SEQ ID NO：35 5′-ACAGTCAGCCGCATCTTCTT-3′
SEQ ID NO：36 5′-ACGACCAAATCCGTTGACTC-3′
SEQ ID NO：37 5′-GTGAGGTCCTCCTTGGTGAA-3′
SEQ ID NO：38 5′-GTGAGGTCCTCCTTGGTGAA-3′
SEQ ID NO：39 5′-TCCCACCTAGAATCTGCTGGC-3′
SEQ ID NO：40 5′-CACATACGGAGCCCCCTTGT-3′
SEQ ID NO：41 5′-GCCAACTGCTTTCATTTGTG-3′
SEQ ID NO：42 5′-ACTCAGGCCCATTTCCTTTA-3′
Embodiment 2：The method for detecting 21 genetic tests of breast carcinoma recurring risk assessment.
Instrument：Roche480 fluorescent quantitative PCR detectors, BECKMAN22R is desk-top micro-
Measure refrigerated centrifuge, Eppendorf 5810R tabletop refrigerated centrifuges, granary Hua Lida laboratory equipments company WH-866 types whirlpool
Revolve oscillator.
(1) paraffin organization RNA extracts reagents
The RNA extraction kit for paraffin-embedded tissue specification that paraffin organization RNA extracting is produced according to Qiagen biotech firms
Extract, obtain tissue RNA standby.
(2) reverse transcription, reagent is purchased from THERMO companies
Reaction system is 20 μ L systems, wherein 11 μ L, RT enzyme of template ribonucleic acid, 1 μ L, RI enzyme 1 μ L, dNTP 2 μ L, 5 × buffer
4 μ L, the μ L of random primer 1；Reaction condition：42 DEG C of 30min, 95 DEG C of 5min.
One sample prepares 3 and managed.
(3) quantitative fluorescent PCR, reagent is purchased from THERMO companies.
Reaction system is 20 μ L, wherein the μ L of 2 × Mix 10, upstream and mixed downstream primer (20 μm of ol/L) 0.4 μ L, template
RNA2 μ L, pyrocarbonic acid diethyl ester (DEPC) processing water 7.6 μ L.
(a) reaction solution is configured：The each component of kit is taken out from -20 DEG C of refrigerators, room temperature is melted, and puts standby on ice chest.Plus
Before sample within 10 minutes, by detection sample number configuration PCR reaction solution X μ L：
X=(the μ L DEPC water of 10 μ L 2 × Mix+0.4 μ L upstream and downstream primers+7.6) × (+1 part of blank control of n parts of samples)
×2。
Pair of primers prepares a pipe overall reaction system, after vibration is mixed, 2000rpm centrifugation 5s, is dispensed by every μ L of person-portion 18
Into 96 hole PCR reaction plates, each sample is repeated 2 times, and reaches sample preparation area standby.
(b) it is loaded：Into the reacting hole for having dispensed reagent, the μ L of template 2 are separately added into, blank control wells add the double steamings of 2 μ L
Water, was loaded range request and operated on ice.Sealed membrane is posted, 3000rpm centrifugation 30s are put into instrument sample introduction tank.
(c) RT-PCR is expanded：Roche480 quantitative real time PCR Instruments carry out breast carcinoma recurring risk simultaneously
21 genetic tests are assessed, reaction condition is as follows：94 DEG C of 5s, 55 DEG C of 35s, carry out 40 circulations, and fluorescent collecting o'clock is at 55 DEG C, reaction
Volume is set to 20 μ L.Preserve file, operation.
(d) interpretation of result, specifically includes following steps：
Roche480 fluorescent PCR detectors click on analysis, and this product belongs to fluoroscopic examination, fluorescence signal
55 DEG C are located at during collection.Quantitative model is chosen, into analysis window, noise line is selected, threshold value is set as just above random
The peak (can suitably be adjusted according to actual conditions) of noise line then, and positive curve is without flex point, it is ensured that under noise line
Numerical value is consistent with the thresholding under analysis item.Experiment blank control wells are as a result qualified without Ct values every time.Click on and calculate, it is automatic to calculate
Obtain the Ct values of each test.
(3) according to breast carcinoma recurring risk assessment formula, the CT values of the sample obtained by step (2) is substituted into formula and are subject to
Calculating a value-at-risk RS, (risk of recurrence scores, from 0 to 100 scope.), judge that recurrence prediction risk is specific according to as follows
Standard：
Low-risk：RS≤18；Risk：18 ＜ RS ＜ 31；Excessive risk：RS≥31
The following is the detection data of 10 samples, while (being purchased from Shanghai Xing Yuan using the genetic test pcr chip of breast cancer 21
Bio tech ltd) compare：
According to criterion, so that it may predict breast carcinoma recurring risk degree, wherein have 3 low-risks, 7 excessive risks.
And it is consistent with genechip detection result.
In summary, the present invention utilizes the advantage of fluorescent quantitation, using fluorescent quantitative poly chain reaction simultaneously to breast
21 genes of gland cancer recurring risk assessment carry out augmentation detection；This kit has simple and efficient to handle, sensitivity and specificity
It is high, stably, it is timely, easy to operate the advantages of, can preferably meet the clinical practice of breast carcinoma recurring risk assessment, it is invalid to reduce
Medication, improves medication accuracy rate, reduces patient economy burden, and extension patient vitals make a contribution.
SEQUENCE LISTING
<110>Hangzhou D.A. Diagnostics Center Co., Ltd. of Wenzhou Di'an Medical Laboratory Institute Co., Ltd.
<120>A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application
<130> SG20170221001
<160> 42
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand cancer related gene Ki67 upstream region of gene primers
<400> 1
cggactttgg gtgcgactt 19
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene Ki67 genes
<400> 2
ttacaactct tccactggga cgat 24
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene STK15 genes
<400> 3
gctagaggca tcatggaccg a 21
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene STK15 genes
<400> 4
gaaggacaca agacccgctg a 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene Survivin genes
<400> 5
agaactggcc cttcttggag g 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene Survivin genes
<400> 6
tctatggggt cgtcatctgg c 21
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene CCNB1 genes
<400> 7
ttcaggttgt tgcaggagac 20
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene CCNB1 genes
<400> 8
catcttcttg ggcacacaat 20
<210> 9
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene MYBL2 genes
<400> 9
gccgagatcg ccaagatg 18
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene MYBL2 genes
<400> 10
cttttgatgg tagagttcca gtgattc 27
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the primer of cancer related gene GRB7 genes
<400> 11
ccatctgcat ccatcttgtt 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the primer of cancer related gene GRB7 genes
<400> 12
ggccaccagg gtattatctg 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene HER2 genes
<400> 13
cggtgtgaga agtgcagcaa 20
<210> 14
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene HER2 genes
<400> 14
cctctcgcaa gtgctccat 19
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> TGTCCAGCCACCAACCAGTG
<400> 15
tgtccagcca ccaaccagtg 20
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene ER genes
<400> 16
ccctcctctt cggtcttttc g 21
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene PgR genes
<400> 17
gcatcaggct gtcattatgg 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene PgR genes
<400> 18
agtagttgtg ctgcccttcc 20
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene BCL2 genes
<400> 19
atcgccctgt ggatgactga g 21
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene BCL2 genes
<400> 20
atgctggggc cgtacagttc 20
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene SCUBE2 genes
<400> 21
tgacaatcag cacacctgca t 21
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene SCUBE2 genes
<400> 22
tgtgactaca gccgtgatcc tta 23
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene MMP11 genes
<400> 23
cccagggcca catttggtt 19
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene MMP11 genes
<400> 24
ggactggctt ttcaccgtcg t 21
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene CTSL2 genes
<400> 25
tgaaagcagt cgcaactgtg g 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene CTSL2 genes
<400> 26
cgaatttgct ccttcaaagc c 21
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene GSTM1 genes
<400> 27
gggacgctcc tgattatgac 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene GSTM1 genes
<400> 28
gtgagcccca tcaatcaagt 20
<210> 29
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene CD68 genes
<400> 29
gcagcacagt ggacattctc g 21
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene CD68 genes
<400> 30
aactgaagct ctgccccagg 20
<210> 31
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of cancer related gene BAG1 genes
<400> 31
cgttgtcagc acttggaata caa 23
<210> 32
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of cancer related gene BAG1 genes
<400> 32
gttcaacctc ttcctgtgga ctgt 24
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of reference gene ACTB genes
<400> 33
ttccttcctg ggcatggagt c 21
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of reference gene ACTB genes
<400> 34
ggtctttgcg gatgtccacg 20
<210> 35
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of reference gene GAPDH genes
<400> 35
acagtcagcc gcatcttctt 20
<210> 36
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of reference gene GAPDH genes
<400> 36
acgaccaaat ccgttgactc 20
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of reference gene RPLPO genes
<400> 37
gtgaggtcct ccttggtgaa 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of reference gene RPLPO genes
<400> 38
gtgaggtcct ccttggtgaa 20
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of reference gene gus gene
<400> 39
tcccacctag aatctgctgg c 21
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of reference gene gus gene
<400> 40
cacatacgga gcccccttgt 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of reference gene TFRC genes
<400> 41
gccaactgct ttcatttgtg 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of reference gene TFRC genes
<400> 42
actcaggccc atttccttta 20
Claims (7)
1. a kind of genetic test primer of breast carcinoma recurring risk assessment 21, it is characterised in that including SEQ ID NO.：1~42 institute
The nucleotide sequence shown.
2. a kind of gene detecting kit of breast carcinoma recurring risk assessment 21, it is characterised in that the kit includes SEQ ID
NO.：Primer shown in 1~42.
3. the genetic test primer of breast carcinoma recurring risk assessment 21 described in claim 1 is in breast carcinoma recurring risk assessment
Using.
4. a kind of gene tester of breast carcinoma recurring risk assessment 21, it is characterised in that comprise the following steps：
(1) paraffin organization RNA is extracted；
(2) reverse transcription；
(3) fluorescent quantitative PCR；
(4) interpretation of result：Judge that recurrence prediction risk is specific according to following standard：Low-risk：RS≤18；Risk：18 ＜ RS
＜ 31；Excessive risk：RS≥31.
5. the gene tester of breast carcinoma recurring risk assessment 21 according to claim 4, it is characterised in that step (2)
In, reverse transcription reaction system is 20 μ L, wherein, 11 μ L, RT enzyme of template ribonucleic acid, 1 μ L, RI enzyme 1 μ L, dNTP 2 μ L, 5 × buffer
4 μ L, the μ L of random primer 1；
Reverse transcription reaction condition：42 DEG C of 30min, 95 DEG C of 5min.
6. the gene tester of breast carcinoma recurring risk assessment 21 according to claim 4, it is characterised in that step (3)
In, fluorescent quantitative PCR system is：PCR reaction systems are 20 μ L, wherein the μ L of 2 × Mix 10, upstream and mixed downstream primer
(10 μm of ol/L) 0.4 μ L, the μ L of template ribonucleic acid 2, pyrocarbonic acid diethyl ester (DEPC) processing water 7.6 μ L.
7. the gene tester of breast carcinoma recurring risk assessment 21 according to claim 4, it is characterised in that step (3)
In, quantitative fluorescent PCR reaction condition is：94 DEG C of 20s, 60 DEG C of 15s, extend 72 degree of 15s, fluorescent collecting o'clock is in 60 DEG C, reactant
Product is set to 20 μ L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710168670.8A CN107058523A (en) | 2017-03-21 | 2017-03-21 | A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710168670.8A CN107058523A (en) | 2017-03-21 | 2017-03-21 | A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107058523A true CN107058523A (en) | 2017-08-18 |
Family
ID=59620850
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710168670.8A Pending CN107058523A (en) | 2017-03-21 | 2017-03-21 | A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058523A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574249A (en) * | 2017-10-23 | 2018-01-12 | 江苏省肿瘤防治研究所（江苏省肿瘤医院） | A kind of expression profiling kit of forecast China crowd breast cancer relapse |
| CN108624697A (en) * | 2018-08-20 | 2018-10-09 | 默禾医疗科技（上海）有限公司 | A kind of 21 gene detecting kit of breast cancer and its detection method |
| CN109439755A (en) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of breast cancer related gene expression |
| CN110129435A (en) * | 2019-02-22 | 2019-08-16 | 深圳塔歌生物技术有限公司 | 21 gene Multiple detection kit of breast cancer |
| CN110777205A (en) * | 2019-11-26 | 2020-02-11 | 合肥仁创基因生物科技有限公司 | Breast cancer 21 gene detection kit and detection method thereof |
| CN110923317A (en) * | 2019-11-27 | 2020-03-27 | 福建省立医院 | Method for breast cancer prognosis prediction and primer group thereof |
| CN112646890A (en) * | 2020-12-29 | 2021-04-13 | 郑鸿钧 | Multi-gene detection primer for predicting distant recurrence risk of early breast cancer |
| CN113436741A (en) * | 2021-07-16 | 2021-09-24 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
| CN113667749A (en) * | 2021-08-03 | 2021-11-19 | 广东省人民医院 | Diagnostic kit for evaluating breast cancer risk by combining four key genes |
| CN113846159A (en) * | 2021-01-27 | 2021-12-28 | 北京百奥纳芯生物科技有限公司 | Special chip for detecting expression of breast cancer related gene |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007061876A2 (en) * | 2005-11-23 | 2007-05-31 | University Of Utah Research Foundation | Methods and compositions involving intrinsic genes |
| US20100222229A1 (en) * | 2003-01-15 | 2010-09-02 | Cobleigh Melody A | Gene Expression Markers for Breast Cancer Prognosis |
| CN102586410A (en) * | 2011-01-18 | 2012-07-18 | 苏州科贝生物技术有限公司 | Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer |
| CN104004844A (en) * | 2014-05-28 | 2014-08-27 | 杭州美中疾病基因研究院有限公司 | Kit for jointly detecting breast cancer 21 genes and preparation method of kit |
-
2017
- 2017-03-21 CN CN201710168670.8A patent/CN107058523A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100222229A1 (en) * | 2003-01-15 | 2010-09-02 | Cobleigh Melody A | Gene Expression Markers for Breast Cancer Prognosis |
| WO2007061876A2 (en) * | 2005-11-23 | 2007-05-31 | University Of Utah Research Foundation | Methods and compositions involving intrinsic genes |
| CN102586410A (en) * | 2011-01-18 | 2012-07-18 | 苏州科贝生物技术有限公司 | Reagent kit for quantitatively assessing long-term recurrence risks of breast cancer |
| CN104004844A (en) * | 2014-05-28 | 2014-08-27 | 杭州美中疾病基因研究院有限公司 | Kit for jointly detecting breast cancer 21 genes and preparation method of kit |
Non-Patent Citations (2)
| Title |
|---|
| PAIK S 等: "A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer", 《N ENGL J MED》 * |
| 李惠 等: "21基因检测对乳腺癌预测和预后的意义", 《现代肿瘤医学》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574249A (en) * | 2017-10-23 | 2018-01-12 | 江苏省肿瘤防治研究所（江苏省肿瘤医院） | A kind of expression profiling kit of forecast China crowd breast cancer relapse |
| CN108624697A (en) * | 2018-08-20 | 2018-10-09 | 默禾医疗科技（上海）有限公司 | A kind of 21 gene detecting kit of breast cancer and its detection method |
| CN108624697B (en) * | 2018-08-20 | 2018-12-18 | 默禾医疗科技（上海）有限公司 | A kind of 21 gene detecting kit of breast cancer and its detection method |
| CN109439755A (en) * | 2018-11-30 | 2019-03-08 | 广东腾飞基因科技股份有限公司 | It is a kind of for detecting the kit of breast cancer related gene expression |
| CN110129435A (en) * | 2019-02-22 | 2019-08-16 | 深圳塔歌生物技术有限公司 | 21 gene Multiple detection kit of breast cancer |
| CN110777205A (en) * | 2019-11-26 | 2020-02-11 | 合肥仁创基因生物科技有限公司 | Breast cancer 21 gene detection kit and detection method thereof |
| CN110923317A (en) * | 2019-11-27 | 2020-03-27 | 福建省立医院 | Method for breast cancer prognosis prediction and primer group thereof |
| CN112646890A (en) * | 2020-12-29 | 2021-04-13 | 郑鸿钧 | Multi-gene detection primer for predicting distant recurrence risk of early breast cancer |
| CN113846159A (en) * | 2021-01-27 | 2021-12-28 | 北京百奥纳芯生物科技有限公司 | Special chip for detecting expression of breast cancer related gene |
| CN113436741A (en) * | 2021-07-16 | 2021-09-24 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
| CN113436741B (en) * | 2021-07-16 | 2023-02-28 | 四川大学华西医院 | Lung cancer recurrence prediction method based on tissue specific enhancer region DNA methylation |
| CN113667749A (en) * | 2021-08-03 | 2021-11-19 | 广东省人民医院 | Diagnostic kit for evaluating breast cancer risk by combining four key genes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107058523A (en) | A kind of genetic test primer of breast carcinoma recurring risk assessment 21 and its application | |
| US11220716B2 (en) | Methods for predicting the prognosis of breast cancer patient | |
| DK2598659T3 (en) | PROCEDURE FOR USING GENEPRESSION FOR DETERMINING PROSTATE CANCER FORECAST | |
| US9249465B2 (en) | Molecular markers for lung and colorectal carcinomas | |
| CN107488740A (en) | Detect the LncRNA combinations of stomach cancer prognosis situation and the kit containing the combination | |
| AU2020204502B2 (en) | Gene expression profile algorithm for calculating a recurrence score for a patient with kidney cancer | |
| WO2009126271A1 (en) | Methods, agents and kits for the detection of cancer | |
| CN110343762A (en) | Bladder carcinoma marker group and its application | |
| US10718030B2 (en) | Methods for predicting effectiveness of chemotherapy for a breast cancer patient | |
| CN105219844A (en) | A kind of compose examination 11 kinds of diseases gene marker combination, test kit and disease risks predictive model | |
| WO2010080702A2 (en) | Gene expression profiling for the identification, monitoring, and treatment of prostate cancer | |
| CN113462776B (en) | m 6 Application of A modification-related combined genome in prediction of immunotherapy efficacy of renal clear cell carcinoma patient | |
| US20110070582A1 (en) | Gene Expression Profiling for Predicting the Response to Immunotherapy and/or the Survivability of Melanoma Subjects | |
| CN107574247A (en) | A kind of the glioblastoma auxiliary diagnosis based on CLCF1 genes, prognostic evaluation kit and its application method | |
| EP4244393A1 (en) | Panel of mirna biomarkers for diagnosis of ovarian cancer, method for in vitro diagnosis of ovarian cancer, uses of panel of mirna biomarkers for in vitro diagnosis of ovarian cancer and test for in vitro diagnosis of ovarian cancer | |
| CN106480201A (en) | Metastasis in Breast Cancer assesses test kit | |
| CN109385474A (en) | Breast cancer molecular parting and DISTANT METASTASES IN risk genes group and diagnostic products and application | |
| CN109097476B (en) | Lung cancer bone metastasis gene detection and diagnosis kit | |
| CN106755330A (en) | Cancer related gene differential expression detection kit and its application | |
| CN108085386B (en) | The identification of the reference gene of osteosarcoma miRNA detection | |
| CN108456730A (en) | Distant place risk of recurrence gene group and in-vitro diagnosis product and application in breast cancer parting | |
| CN108929915B (en) | Lung cancer bone metastasis gene detection and diagnosis kit | |
| CN108949999B (en) | Gene detection kit for diagnosing lung cancer bone metastasis | |
| WO2010062763A1 (en) | Gene expression profiling for predicting the survivability of melanoma subjects | |
| CN113278702A (en) | Application of PSMC2 gene detection primer in preparation of glioblastoma multiforme auxiliary diagnosis and prognosis evaluation kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170818 |