CN109609646A - It is a kind of for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility - Google Patents
It is a kind of for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility Download PDFInfo
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Abstract
The invention discloses a kind of for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility, belongs to biology techniques field.The kit includes two pairs of Specific PCR primers based on the design of NFE2L2 gene mononucleotide polymorphism.There are single nucleotide polymorphism: T > G by the 522nd of nucleotide sequence molecular labeling as shown in SEQ ID NO.1, that is the 522nd base of this sequence includes two kinds of situations of base T and bases G, corresponding gene type is TT, tri- kinds of types of TG and GG, with ovarian epithelial carcinoma neurological susceptibility close association, the single nucleotide polymorphism in NFE2L2 gene effectively can be quickly detected using round pcr, and then predict to suffer from the neurological susceptibility of ovarian epithelial carcinoma, this screening and monitoring for ovarian epithelial carcinoma Susceptible population, early warning is carried out to ovarian epithelial carcinoma, early diagnosis and individualized treatment have important value for clinical application.
Description
Technical field
The present invention relates to biology techniques fields, and in particular to a kind of for monitoring the molecule of ovarian epithelial carcinoma neurological susceptibility
Label and its clinical application.
Background technique
Oophoroma is a kind of female malignant of high mortality, and the number that ovarian epithelial carcinoma is died of in the whole world every year is super
Cross 150,000 people.Ovarian cancer patients disease progression is rapid, and when patient assessment often has evolved to late stage, intraperitoneal to turn extensively
It moves and involves multiple peritonaeum organs, most of early ovarian cancers do not have any clinical symptoms, up to the present without fine yet
Early prediction index, the early warning of early stage can not be carried out.
The treatment of oophoroma includes the combined chemotherapy based on surgical cytoreduction and platinum class, although 80% oophoroma is suffered from
Person initially has a significant reaction to this comprehensive therapeutic plan, but most of can all recur after drug resistance.In the U.S., cure rate from
The 12% of the 1970's only only increases to 14% in 2000.And 5 years survival rates of early ovarian cancer patient can be up to 90%, this
If us is prompted to find early monitoring index, early prediction, early diagnosis and early treatment are carried out to oophoroma Susceptible population
The survival rate that oophoroma can be increased substantially, finally improves prognosis.
The generation of oophoroma and the genetic background of individual are closely related, have apparent heredity.Cancer gene group map
(TCGA) planning studies are shown, there are the excessive amplifications of more than 30 a growth stimulation genes for 10% advanced ovarian cancer case, and
It is related with base mutation with the abnormal recombination of BRCA1/2 gene with TP53 gene, it is also possible to and the nucleotide variation of some genes has
It closes.Therefore the genetic molecule label for finding detection oophoroma Susceptible population, disease control and treatment to oophoroma have important
Prevention and clinical meaning.But these current Research on Genetic Variation cannot explain the genetic background and disease of ovarian epithelial carcinoma completely
The neurological susceptibility of disease.Therefore, it is necessary to more extensive genetic background research, the heredity back for comprehensively explaining ovarian epithelial carcinoma is made every effort to
Scape, while preferably disease effectively can be monitored with molecular marked compound.
Gene is also referred to as NRF2 to NFE2L2 (Nuclear factor erythroid 2-like 2), is cell to oxidation
With it is electrophilic stress protection mechanism key transcription factor.Document report shows as high expression in kinds of tumors, including lung cancer,
Breast cancer and colon cancer, highly expressed NFE2L2 albumen are thin in tumour by improving active oxygen inhibition enzyme and drug outflow transporter
Key effect is played in intracellular growth and chemotherapy resistance, can regard one kind of tumor marker as, but due to being protein level
Detection, which can not carry out effective early prediction and diagnosis, the change if that can detect genetic background to disease, can accomplish to disease
Early warning and monitoring, but up to the present without document report NFE2L2 gene DNA level single nucleotide polymorphism
(SNP) relationship between variation and the neurological susceptibility of tumour.
Summary of the invention
The present invention is intended to provide a kind of develop use for monitoring the molecular labeling of ovarian epithelial carcinoma neurological susceptibility, and with this
In the kit of monitoring ovarian epithelial carcinoma neurological susceptibility, for discovery ovarian epithelial carcinoma Susceptible population and epithelial ovarian carcinoma patients
It carries out effectively early diagnosing the detection method provided with personalized treatment effectively with sensitivity.
To achieve the above object, the present invention adopts the following technical scheme:
Nucleotide sequence science of heredity molecular labeling as shown in SEQ ID NO.1 monitors epithelium in preparation as detection target spot
Application in the detection kit of property susceptibility of ovarian cancer, the 522nd of the science of heredity molecular labeling that there are mononucleotides is more
State property: T > G, the i.e. base of the 522nd of this sequence include two kinds of situations of base T and bases G, corresponding gene type be TT, TG and
Tri- kinds of types of GG.
It is normal NFE2L2 gene genetic state when showing as base T, is epithelial ovarian when showing as bases G
The susceptible NFE2L2 gene genetic state of cancer.
The hair of certain mononucleotide polymorphism sites and oophoroma on early-stage study discovery tumor marker genes of the present invention
Existence is reported the single nucleotide polymorphism of NFE2L2 gene specific site and to suffer from ovarian epithelial carcinoma close for the first time in correlation
Correlation, result of study show that the site 522T > G and ovarian epithelial carcinoma neurological susceptibility in SEQ ID NO.1 (rs17524059) are deposited
It is significantly being associated with, is showing as 522GG homozygote genotype individual and suffer from the risk of ovarian epithelial carcinoma significantly improving, therefore can be with
The molecular labeling for monitoring and early diagnosing as ovarian epithelial carcinoma neurological susceptibility.
As the application of above-mentioned molecular labeling, the present invention provides a kind of for monitoring the inspection of ovarian epithelial carcinoma neurological susceptibility
Test agent box, comprising: specific amplification nucleotide sequence is the 522nd in the science of heredity molecular labeling as shown in SEQ ID NO.1
SNP site primer pair.
For the single nucleotide polymorphism of above-mentioned NFE2L2 gene specific site, the present invention has designed and synthesized one specifically
The downstream PCR primer and two specific upstream PCR primers, each specific forward primer of property respectively correspond corresponding T, G
Base sequence makees PCR amplification, product length 174bp, according to the PCR sun of corresponding pairing primer amplification with downstream primer respectively
Property band judge that the genotype of corresponding base is homozygote or heterozygote.
Simultaneously in order to improve the specificity that specific base detects, 3 ' ends of upstream primer are fallen when swimming primer in design
Second base of number is designed as base identical with complementary strand, rather than complementary base, this can preferably be avoided non-specificity
The appearance of PCR amplification band ensure that the high degree of specificity of testing result.
Specifically, the PCR upstream and downstream primer sequence are as follows:
Upstream primer 1:5 '-CCGCGCCCACAGACGGCCGC-3 ' (SEQ ID NO.2);
Upstream primer 2:5 '-CCGCGCCCACAGACGGCCGA-3 ' (SEQ ID NO.3);
Downstream primer: 5 '-GCTGCGCGGCTCCCCCGCCT-3 ' (SEQ ID NO.4).
The kit also includes PCR reaction solution, and the PCR reaction solution includes PCR buffer, dNTP mixed liquor and Taq
Archaeal dna polymerase.Dose relationship in PCR reaction solution between each component is the ratio under the conditions of Standard PCR, to fields technology
Personnel are Conventional wisdoms.
PCR reaction system composition includes: to contain Mg in terms of 20 μ L of total volume2+10 × PCR buffer, 2 μ L, 2.5mM's
Draw in the downstream of upstream primer 1 the μ L, 100pmol of Taq archaeal dna polymerase 0.2 the μ L, 100pmol of 8 μ L, 5U/ μ L of dNTP mixed liquor
1 μ L of object.
The detection kit further includes positive control I and positive control II, and the positive control I contains nucleotide sequence
The genetic fragment as shown in SEQ ID NO.5;The positive control II contains nucleotide sequence base as shown in SEQ ID NO.6
Because of segment.
As the application of mentioned reagent box, the present invention also provides a kind of external DNA samples of detection whether there is NFE2L2
The method of the single nucleotide polymorphism of gene, the specific steps are as follows:
(1) genomic DNA in peripheral blood cells is extracted as template, utilizes NFE2L2 gene specific upstream and downstream primer
PCR amplification is carried out, amplified production is obtained, the NFE2L2 Gene specific PCR primers are two pairs, and nucleotide sequence is respectively
SEQ ID NO.2 and SEQ ID NO.4, SEQ ID NO.3 and SEQ ID NO.4.
(2) agarose gel electrophoresis detects pcr amplification product positive band, according to as a result, judging that NFE2L2 gene is specific
Site whether there is following single nucleotide polymorphism: 522T > G, and polymorphism position number is based on SEQ ID NO.1 base sequence
Column, so that it is determined that the NFE2L2 gene specific site in peripheral blood cells whether there is single nucleotide polymorphism.
Risk prediction and diagnosis are carried out to ovarian epithelial carcinoma neurological susceptibility individual using kit provided by the invention
When, it is compared with normal NFE2L2 gene base: the 522T > G that such as has differences, that is, there is single nucleotide polymorphism is then said
The risk that the bright individual suffers from ovarian epithelial carcinoma is higher than normal population.
It is that the present invention has the utility model has the advantages that
The present invention provides a kind of for monitoring the detection kit of epithelial ovarian genetic susceptibility of cancer, contains in kit
There are two pairs of Specific PCR primers based on NFE2L2 gene mononucleotide polymorphism site rs17524059 design, NFE2L2 base
It, can be efficiently special using specific PCR amplification technique because single nucleotide polymorphism is significantly associated with ovarian epithelial carcinoma neurological susceptibility
The single nucleotide polymorphism of different detection NFE2L2 gene specific site, this method is fast and convenient and cost performance and specificity are high,
Without special DNA sequencing equipment, convenient for being promoted and applied in basic hospital, this prison for ovarian epithelial carcinoma Susceptible population
It surveys, carrying out early warning, diagnosis and individualized treatment to ovarian epithelial carcinoma has important clinical meaning.
Specific embodiment
It elaborates below with reference to embodiment to the present invention.The purpose of method used in the examples below is preferably
Understand the present invention, but is not limited to the present invention.
Unless otherwise specified, experimental method involved in embodiment is conventional method, and experiment reagent used is normal
Advise Reagent Company's purchase.
The association analysis of embodiment 1.NFE2L2 gene mononucleotide polymorphism detection and ovarian epithelial carcinoma neurological susceptibility
1.1 research object
Case access time is in July, 2007 in December, 2012, comes from Hospital for Gynaecology and Obstetrics Attached to Medical Hospital of Zhejiang
The specific case of pathological diagnosis randomly selects ovarian epithelial carcinoma case 136, while choosing 325 normal physical examinations of same time
Volunteer (women) make normal control, the inclusion criteria of normal control is or without other gynecological tumors and without other solid carcinomas exempt from
Epidemic disease disease.All cases and normal control individuals are Donors.All subjects are according to Medical College of Zhejiang Univ.
Ethics Committee, hospital for obstetrics and gynaecology requires informed consent, and all research methods have followed the guilding principle and regulations of approval.
Patient and Healthy Volunteers periphery anticoagulation 2ml or so are extracted, it is stand-by in -80 DEG C of freezen protectives.
1.2 peripheral blood cells genome DNA extractions
To 100 microlitres of peripheral bloods carry out genomic DNA extracting and purifying (Shanghai Sheng Gong bioengineering Co., Ltd it is outer
All blood extraction agent boxes), experiment illustrates to carry out according to the step of kit, extracts obtained genomic DNA and is dissolved in deionized water
And cryogenic freezing saves in case subsequent PCR detection uses.
1.3PCR reaction solution composition, specific upstream and downstream primer and PCR condition
The base sequence (rs17524059) of NFE2L2 gene is downloaded from the single nucleotide polymorphism library (SNP) of GenBank,
And design of primers is carried out with online primer-design software Primer-BLAST, in order to further increase the special of detection single base
Property, the base of the end of upstream primer 3 ' penultimate is changed to according to our own design principle and experience identical as complementary strand
Base, finally determine our self-designed upstream primers, specific primer base sequences are shown in Table 1.
1 NFE2L2 single nucleotide polymorphism of table detection upstream and downstream primer and product length
PCR reaction solution composition: totally 20 μ L total volume includes: PCR buffer, 0.25mM dNTP, 1.0U Taq DNA
Polymerase, each 5.0pmol of upstream and downstream primer, 15ng genomic DNA.
Pcr amplification reaction condition: 94 DEG C 5 minutes, (94 DEG C 30 seconds, 56.5 DEG C 35 seconds, 72 DEG C 30 seconds) × 40 circulations, 72
DEG C extend 10 minutes, 10 DEG C heat preservation, product length 174bp.
Pcr amplification product detection: 10 μ LPCR amplified productions are added 2.0% Ago-Gel and are separated by electrophoresis, 50V electricity
Piezoelectricity is swum 20 minutes, ethidium bromide (EB) dyeing, observes positive band under ultraviolet lamp.The PCR product matched according to different primers
Electrophoretic band analyzes the nucleotide polymorphism implementations of 522 site of NFE2L2 gene (position number is based on SEQ ID NO.1).
Through experimental tests and statistical analysis, it has been found that there are mononucleotide as 522T > G is more in institute's sample sheet
State property shows as three kinds of TT homozygote, TG heterozygote and GG homozygote genotype.
The association analysis of 1.4NFE2L2 gene mononucleotide polymorphism genotype and ovarian epithelial carcinoma
It is with reference to the high-risk correlation analysis of progress with Normal group.It is single to NFE2L2 gene specific site (rs17524059)
The genotype and ovarian epithelial carcinoma risk of nucleotide polymorphisms carry out correlation analysis, are returned by binary logistic
Analysis obtains odds ratio (Odds ratio, OR), 95% confidence interval (confidence interval, CI) and P value.It is all
Statistics is two-sided criterion of significance, and significance is set as P value less than or equal to 0.05.All statistical analysis are all made of
SPSS18.0 software (SPSS Inc.Chicago, USA).
As a result, it has been found that there are significant phases with ovarian epithelial carcinoma is suffered from by 522T > G in SEQ ID NO.1 (rs17524059)
Guan Xing, 522GG homozygote significantly improve the risk that individual suffers from ovarian epithelial carcinoma, and OR value has reached 3.15 (1.50-
6.61);522G gene frequency value-at-risk OR has reached 1.60 (1.15-2.21), hence it is evident that is higher than Normal group, specific number
According to being shown in Table 2.
Table 2.NFE2L2 gene nucleotide variation association analysis and the risk for suffering from ovarian epithelial carcinoma
Embodiment 2.NFE2L2 gene mononucleotide polymorphism and ovarian epithelial carcinoma neurological susceptibility detection kit
1, the result based on embodiment 1, it has been found that the base of 522T > G in SEQ ID NO.1 (rs17524059) becomes
It is different to suffer from ovarian epithelial carcinoma there are significant related, therefore we devise for NFE2L2 gene specific site
(rs17524059) the specific PCR upstream and downstream primer that single nucleotide polymorphism is detected, final design is to Susceptible population
The kit that is detected of peripheral blood DNA template.
2, it prepares dedicated kit (100tests), specific reagent set is as follows at being shown in Table 3:
Table 3.NFE2L2 gene mononucleotide polymorphism site primer kit reagent is at being grouped as
Reagent name (concentration) | Kit total amount |
10×PCR Buffer(Mg2+25mM) | 200μL |
DNTP Mixture (each 2.5mM) | 800μL |
Taq DNA polymerase(5U/μL) | 20μL |
Upstream primer 1 (SEQ ID NO.2) (100pmol) | 100μL |
Upstream primer 2 (SEQ ID NO.3) (100pmol) | 100μL |
Downstream primer (SEQ ID NO.4) (100pmol) | 100μL |
Positive control I (SEQ ID NO.5) | 10μL |
Positive control II (SEQ ID NO.6) | 10μL |
3, prepared by peripheral blood cells genome DNA extraction: subject's peripheral blood 1mL is extracted, using conventional phenol chloroform
Method or third party's peripheral blood cells genome DNA extraction kit, the genomic DNA extracted are dissolved in deionized water,
Cryo-conservation is stand-by.
4, PCR reagent formula is individually detected, is specifically shown in Table 4:
Table 4
10×PCR Buffer(Mg2+25mM) | 2μL |
DNTP Mixture (each 2.5mM) | 8μL |
Taq DNA polymerase(5U/μL) | 0.2μL |
Upstream primer (100pmol) | 1 μ L (optional one) in 1,2 |
Downstream primer (100pmol) | 1μL |
Total volume | 20μL |
5, pcr amplification reaction condition:
PCR reaction solution composition: totally 20 μ L total volume includes: PCR buffer, 0.25mM dNTP, 1.0U Taq DNA
Polymerase, each 5.0pmol of upstream and downstream primer, 15ng genomic DNA.
Pcr amplification reaction condition: 94 DEG C 5 minutes, (94 DEG C 30 seconds, 56.5 DEG C 35 seconds, 72 DEG C 30 seconds) × 40 circulations, 72
DEG C extend 10 minutes, 10 DEG C heat preservation, product length 174bp.
6, pcr amplification product is observed: 10 μ LPCR amplified productions are added 2.0% Ago-Gel and are separated by electrophoresis, 50V
Electrophoresis 20 minutes, ethidium bromide (EB) dyed, and observed positive band under ultraviolet lamp.It is produced according to the PCR of different primers pairing
Object electrophoretic band analyzes the nucleotide polymorphism implementations of 522 site of NFE2L2 gene (position number is based on SEQ ID NO.1).
It is compared with normal NFE2L2 gene base sequence, has differences and show that the individual suffers from epithelial ovarian
The risk of cancer is higher than normal population.I.e. there are single nucleotide polymorphism: 522T > G for the difference.
Applicant's statement: after reading above content of the invention, in specific implementation detection, the present invention is done various
Change or finishing, equivalent form replacement for example of the invention are also fallen within protection scope of the present invention.
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<223> n is a, c, g, t or u
<400> 1
gctgtggagc tagggaaccg cggcccggga gggacagccg caaccggctt cccccacgtc 60
tggccagagc caggaccgcg gcgctgggta gagccgccgc gcttgcgccg gggcagggcg 120
gggaggggca gcggggacgc ggccgggtga tccgaccgac cacgagcccg agggcgaacg 180
ggtgggaagt tgcgggaagg tctggggact gagcccgctc gcgtgggcct tgggggagaa 240
tccagccgcg tccccgggcc cgagagctgg gactccggag cccctaagtt tgagcggccc 300
ggtgggcggc ggggcaagag ggggcggacg ctggccgtct gagccggcgc ggcccggccc 360
ttccggggct gcgcggctcc cccgcctcgg tgccggcaaa aatgtgccta gtcacggggc 420
cgctctcggg ggaactgagg tcgccttcgg gctgggaccc ggagcccctt cgccgcgccc 480
caagacctcc ttgagtgcgg gctgcgacgc gctcaccccg cngggccgtc tgtgggcgcg 540
gctttgcgaa gtcatccatc tctcggatca ctctctggca gccttgagct ctcttgaaag 600
cccagcccc 609
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccgcgcccac agacggccgc 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ccgcgcccac agacggccga 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gctgcgcggc tcccccgcct 20
<210> 5
<211> 174
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gctgcgcggc tcccccgcct cggtgccggc aaaaatgtgc ctagtcacgg ggccgctctc 60
gggggaactg aggtcgcctt cgggctggga cccggagccc cttcgccgcg ccccaagacc 120
tccttgagtg cgggctgcga cgcgctcacc ccgctgggcc gtctgtgggc gcgg 174
<210> 6
<211> 174
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gctgcgcggc tcccccgcct cggtgccggc aaaaatgtgc ctagtcacgg ggccgctctc 60
gggggaactg aggtcgcctt cgggctggga cccggagccc cttcgccgcg ccccaagacc 120
tccttgagtg cgggctgcga cgcgctcacc ccgcggggcc gtctgtgggc gcgg 174
Claims (6)
1. nucleotide sequence science of heredity molecular labeling as shown in SEQ ID NO.1 is epithelial in preparation monitoring as detection target spot
Application in the detection kit of susceptibility of ovarian cancer, which is characterized in that the 522nd of the science of heredity molecular labeling exists single
Nucleotide polymorphisms: T > G, the i.e. base of the 522nd of this sequence include two kinds of situations of base T and bases G.
2. a kind of for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility characterized by comprising specific amplification core
The primer pair of nucleotide sequence the 522nd SNP site in the science of heredity molecular labeling as shown in SEQ ID NO.1.
3. as claimed in claim 2 for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility, which is characterized in that described
Primer pair be two pairs, the nucleotide sequence of the upstream primer of one of primer pair as shown in SEQ ID NO.2, downstream primer
Nucleotide sequence is as shown in SEQ ID NO.4, the nucleotide sequence of the upstream primer of another primer pair such as SEQ ID NO.3 institute
Show, the nucleotide sequence of downstream primer is as shown in SEQ ID NO.4.
4. as claimed in claim 2 for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility, which is characterized in that also wrap
PCR reaction solution is included, the PCR reaction solution includes dNTP mixed liquor, Taq archaeal dna polymerase and PCR buffer.
5. as claimed in claim 4 for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility, PCR reaction system composition
It include: to contain Mg in terms of 20 μ L of total volume2+10 × PCR buffer 2 μ L, 2.5mM 8 μ L, 5U/ μ L of dNTP mixed liquor Taq
The 1 μ L of downstream primer of upstream primer 1 the μ L, 100pmol of archaeal dna polymerase 0.2 μ L, 100pmol.
6. as claimed in claim 2 for monitoring the detection kit of ovarian epithelial carcinoma neurological susceptibility, which is characterized in that also wrap
Positive control I and positive control II are included, the positive control I contains nucleotide sequence gene piece as shown in SEQ ID NO.5
Section;The positive control II contains nucleotide sequence genetic fragment as shown in SEQ ID NO.6.
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CN105779434A (en) * | 2014-12-15 | 2016-07-20 | 天津华大基因科技有限公司 | Kit and applications thereof |
CN104962655A (en) * | 2015-07-28 | 2015-10-07 | 山东大学齐鲁医院 | Ovarian cancer susceptibility-related molecular marker as well as detection primer and kit |
WO2018009939A1 (en) * | 2016-07-08 | 2018-01-11 | Genentech, Inc. | Methods for diagnosing and treating cancer by means of the expression status and mutational status of nrf2 and downstream target genes of said gene. |
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PANAGIOTIS A. KONSTANTINOPOULOS等: ""Keap1 Mutations and Nrf2 Pathway Activation in Epithelial Ovarian Cancer"", 《CANCER RESEARCH》 * |
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