CN108456723A - A kind of the genetic test primer and kit of endometriosis risk profile - Google Patents
A kind of the genetic test primer and kit of endometriosis risk profile Download PDFInfo
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Abstract
The present invention a kind of the genetic test primer and kit of endometriosis risk profile, belong to biomedicine technical field.Primer includes the amplimer of the PCR amplification primer for 24 SNP sites for detecting human genome, sequencing library structure.Kit includes above-mentioned primer, PCR premixed liquids and PCR product purifying magnetic bead.The present invention be directed to the designs of the susceptibility loci of Chinese population endometriosis, and in conjunction with multiplying property risk evaluation model, Endometriosis incidence risk can be effectively predicted.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of gene inspection of endometriosis risk profile
Survey primer, kit and its application.
Background technology
Endometriosis (endometriosis, EMs) refers to endometrial gland and interstitial with growth function
Other positions other than uterine cavity lining mucosa are grown in, ectopic growth, development, bleeding cause symptom, be mainly characterized by basin
Chamber pain, mass and infertile.It is a kind of common disease of the women of child-bearing age, and incidence has ascendant trend year by year, drastically influences
The Health and Living quality of young women causes huge financial burden for society., the only U.S. in 2002, with gynecopathy phase
The various medical expenses closed are up to 22,000,000,000 dollars.And in China, average time in hospital of the gynecopathy patient between 15 years 1994 to 2008 years
Expense increases to 7853 yuan by 3382 yuan, this number is also in being continuously increased.
However the occurrence and development process of endometriosis is still unsolved mystery, from endometrial implantation theory, in place
Inner membrance determines theory, theory Of heredity, immunological theory, stem cell theory etc., and any type theory cannot all explain all gynecopathies
Morbidity, just as its various clinical symptoms and pathological characters.EMs is typically a slow process, in addition to one
Also have outside a little environmental factors and carry the inherent causes such as endometriosis risk genes, is gene and the coefficient knot of environment
Fruit.
By genetic test and genetic counselling, the onset risk of some diseases can be predicted, it is thus understood that whether is risk genes
The next generation can be entailed, disease prevention means management individual health is can be combined with.Currently, the genetic test in China and heredity are consulted
It askes also in the early stage of development, the disease prevention of early stage is often ignored, and more people were just felt in morbidity mid-term even late period
It observes.By genetic test, Endometriosis incidence risk is predicted, to realize that morning is known, morning prevents.
Due to heterogeneity, the pleiotropism etc. of heredity, such as disease-susceptible humans site, the susceptibility loci frequency of mutation, disease
Incidence has different degrees of difference in different populations, particularly, is studied from whole-genome association
(GWAS) requirement of the disease-susceptible humans site to population is even more stringent, these factors promote disease risks predicted gene to detect needs
It is optimized according to the hereditary feature of Chinese population.
Invention content
For this reason, it may be necessary to provide a kind of genetic test primer and kit for endometriosis risk profile, needle
Endometrium is predicted in conjunction with two generation sequencing technologies and bioinformatic analysis method to the susceptibility loci of endometriosis
Endometriosis onset risk.
The genetic test primer of endometriosis risk profile, including 24 SNP sites of detection human genome
PCR amplification primer.
24 SNP sites be rs12024204, rs801112, rs16826658, rs7521902,
rs10508881、rs11193561、rs10431397、rs3596、rs10859871、rs1268843、rs12449465、
rs4141819、rs1250248、rs13394619、rs6757804、rs1519761、rs6907340、rs7739264、
rs12700667、rs2270221、rs1333049、rs10975519、rs1537377、rs10965235。
Wherein, the PCR amplification primer sequence in the sites rs12024204 is respectively SEQ ID NO：1 and SEQ ID NO：25；
The PCR amplification primer sequence in the sites rs801112 is respectively SEQ ID NO：2 and SEQ ID NO：26；
The PCR amplification primer sequence in the sites rs16826658 is respectively SEQ ID NO：3 and SEQ ID NO：27；
The PCR amplification primer sequence in the sites rs7521902 is respectively SEQ ID NO：4 and SEQ ID NO：28；
The PCR amplification primer sequence in the sites rs10508881 is respectively SEQ ID NO：5 and SEQ ID NO：29；
The PCR amplification primer sequence in the sites rs11193561 is respectively SEQ ID NO：6 and SEQ ID NO：30；
The PCR amplification primer sequence in the sites rs10431397 is respectively SEQ ID NO：7 and SEQ ID NO：31；
The PCR amplification primer sequence in the sites rs3596 is respectively SEQ ID NO：8 and SEQ ID NO：32；
The PCR amplification primer sequence in the sites rs10859871 is respectively SEQ ID NO：9 and SEQ ID NO：33；
The PCR amplification primer sequence in the sites rs1268843 is respectively SEQ ID NO：10 and SEQ ID NO：34；
The PCR amplification primer sequence in the sites rs12449465 is respectively SEQ ID NO：11 and SEQ ID NO：35；
The PCR amplification primer sequence in the sites rs4141819 is respectively SEQ ID NO：12 and SEQ ID NO：36；
The PCR amplification primer sequence in the sites rs1250248 is respectively SEQ ID NO：13 and SEQ ID NO：37；
The PCR amplification primer sequence in the sites rs13394619 is respectively SEQ ID NO：14 and SEQ ID NO：38；
The PCR amplification primer sequence in the sites rs6757804 is respectively SEQ ID NO：15 and SEQ ID NO：39；
The PCR amplification primer sequence in the sites rs1519761 is respectively SEQ ID NO：16 and SEQ ID NO：40；
The PCR amplification primer sequence in the sites rs6907340 is respectively SEQ ID NO：17 and SEQ ID NO：41；
The PCR amplification primer sequence in the sites rs7739264 is respectively SEQ ID NO：18 and SEQ ID NO：42；
The PCR amplification primer sequence in the sites rs12700667 is respectively SEQ ID NO：19 and SEQ ID NO：43；
The PCR amplification primer sequence in the sites rs2270221 is respectively SEQ ID NO：20 and SEQ ID NO：44；
The PCR amplification primer sequence in the sites rs1333049 is respectively SEQ ID NO：21 and SEQ ID NO：45；
The PCR amplification primer sequence in the sites rs10975519 is respectively SEQ ID NO：22 and SEQ ID NO：46；
The PCR amplification primer sequence in the sites rs1537377 is respectively SEQ ID NO：23 and SEQ ID NO：47；
The PCR amplification primer sequence in the sites rs10965235 is respectively SEQ ID NO：24 and SEQ ID NO：48.
The genetic test primer of endometriosis risk profile further includes the amplimer of sequencing library structure.
The amplimer of the sequencing library structure includes 1 positive universal primer and 12 reverse primers, and forward direction is logical
It is SEQ ID NO with the sequence of primer：49, reverse primer sequences are SEQ ID NO：50-61.
The gene detecting kit of endometriosis risk profile includes 24 SNP sites of detection human genome
PCR amplification primer, sequencing library structure amplimer, PCR premixed liquids, PCR product purify magnetic bead.
Wherein, PCR premixed liquids include PCR buffer solutions, magnesium ion, dNTP mixtures, archaeal dna polymerase and water.
Further, the gene detecting kit of endometriosis risk profile is in Endometriosis incidence wind
Application in the prediction of danger.
Scheme as a further improvement on the present invention, mentioned reagent box further include the alkali for being handled PCR product
Acid phosphatase.
It is different from the prior art, the advantages of above-mentioned technical proposal is：
(1) present invention aims at Chinese's design, is directed to the susceptibility loci of Chinese population endometriosis,
In conjunction with multiplying property risk evaluation model, Endometriosis incidence risk can be effectively predicted.
(2) present invention is on the basis of the achievement of forefathers studied for endometriosis tumor susceptibility gene, for son
24 mononucleotide polymorphism sites of Endometriosis onset risk are designed to a panel, utilize newest amplicon
Sequencing technologies obtain the information limited in the past without that can obtain due to researcher's self-technique and knowledge.
Description of the drawings
Fig. 1 is that sample mixes electrophoresis result, and wherein M is 1Kb marker, and band molecular weight from top to bottom is respectively
100,200,300,400,500,650,850,1000,1650,2000,3000,4000bp, 1 swimming lane is mixing before the sequencing of upper machine
The electrophoretic band in library.
Fig. 2 is endometriosis risk forecast model AUC curve graphs.
Fig. 3 is distributed for reads base sequencing qualities.
Fig. 4 is the target area mean coverage of filtered quality data.
Fig. 5 is the median of sample overburden depth.
Specific implementation mode
For the technology contents of technical solution, construction feature, the objects and the effects are described in detail, below in conjunction with specific reality
It applies example and attached drawing is coordinated to be explained in detail.
The present invention uses the disease-susceptible humans site that Chinese population is directed in whole-genome association research (GWAS), screening
Go out to be effectively predicted 24 mononucleotide polymorphism sites (SNP) of Endometriosis incidence risk；According to these
SNP, the primer sets that design is reacted for site pcr amplification reaction and amplified library accordingly.Acquire subject saliva or
Blood sample extracts DNA；Target gene is amplified by multi-PRC reaction, then is connect plus sequencing by library primer amplification
Head is then sequenced by MiSeq and generates data；Sequencing data is analyzed by raw letter, is generated genotypic results, is used multiplying property
Risk model (multiplicative risk model) analyzes subject's Endometriosis incidence risk, and then can be with
According to prediction result, scientific and effective individual health governing plan is formulated, reduces the influence that high risk factor is brought.
By screening the endometriosis susceptibility loci in GWAS, the genotype of these SNP is detected, in conjunction with mathematics point
Model is analysed, predicts the onset risk of endometriosis.Step summary is as follows：
1. screening the disease-susceptible humans site in GWAS researchs
Search is based on Chinese population on the document retrieval system (PubMed) of American National Bioinformatics Institute (NCBI)
GWAS researchs in endometriosis susceptibility loci, while retrieving the database that GWAS Catalog are provided, filter out
In GWAS researchs positive sample size be more than 750, correct after P values<0.01, it is tested in extensive case-control group research
The susceptibility loci of card, and these sites frequency in Chinese population is required to be not less than 0.05, if the chain injustice between SNPs
Weigh coefficient r2>0.8, then choose odds ratio (OR) maximum SNP.
The endometriosis susceptibility loci obtained after screening, as shown in table 1.
The endometriosis susceptibility loci that table 1 screens
2. design of primers and synthesis
The PCR amplification primer of SNP site to be measured, and the amplimer of synthetic library structure are designed and synthesized, for surveying
Sequence.SNP site and corresponding PCR amplification primer are shown in Table 2, and the amplimer of library construction is shown in Table 3.
The sites table 2SNP and corresponding PCR amplification primer
The amplimer of 3 library construction of table
3. extracting genomic DNA in people's saliva or blood
Using Hipure Blood DNA Mini Kits (Magen, D3111) or similar products, people's saliva or blood are extracted
DNA in liquid.Quantitative with spectrophotometer, agarose gel electrophoresis quality inspection, genome dna electrophoresis band is usually not less than
10kb.The DNA concentration of quality inspection qualification is diluted to 10ng/ μ L, 20 DEG C store for future use.
4. site PCR amplification
It using multiple PCR technique, is carried out in 0.2mL PCR pipes, each reaction system total volume is 20 μ L.
(1) concentration synthesized according to SEQ ID every primer of NO.1-48, the multiplex PCR for being mixed into every 5 μM of primer draw
Object mixture.
(2) PCR reaction system mixed liquors are prepared in 0.2mL PCR pipes, one, each sample reacts, as shown in table 4.
4 site PCR reaction system mixed liquors of table
| PCR reaction systems | To each reaction (μ L) |
| 2X PCR premixed liquids | 10 |
| PCR primer mixture (each 5 μM) | 2 |
| Genomic DNA solution (10ng/ μ L) | 2 |
| Water | 6 |
| Total volume | 20 |
(3) set in PCR instrument PCR reaction conditions as：95 DEG C 10 minutes；95 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds,
25 cycles；72 DEG C 3 minutes；12 DEG C of holdings.PCR pipe is put in PCR instrument, PCR reactions are started.
(4) after PCR reactions, PCR is produced using 1X (20 μ L) PCR product purifying magnetic bead (Beaverbio, 70401-100)
Object is purified, and carries out being purified by flash product using 40 μ L ultra-pure waters.
5. Library PCR amplification
Using secondary PCR technology, sequence measuring joints sequence is added for the PCR product of the SNP containing site.In 0.2mL PCR pipes
It carries out, each reaction system total volume is 20 μ L.
(1) PCR reaction system mixed liquors are prepared in 0.2mL PCR pipes, one, each sample reaction, using different anti-
To primer, as shown in table 5.
5 library PCR reaction system mixed liquors of table
| PCR reaction systems | To each reaction (μ L) |
| 2X PCR premixed liquids | 25 |
| Positive universal primer (10 μM) | 2.5 |
| Reverse primer X (10 μM, 1-12 is any) | 2.5 |
| Upper step PCR product | 20 |
| Total volume | 50 |
(2) set in PCR instrument PCR reaction conditions as：95 DEG C 10 minutes；95 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8
A cycle；72 DEG C 3 minutes；12 DEG C of holdings.PCR pipe is put in PCR instrument, PCR reactions are started.
(3) after PCR reactions, PCR product is purified using 1X (50 μ L) PCR purifying magnetic beads, and ultrapure using 25 μ L
Water be purified by flash after library.
6. library Quality Control
(1) it carries out qBit to library to quantify, it is ensured that each library is up-to-standard.
(2) according to quantitative result, sample of the mixed in equal amounts containing different labels.
It selects 100 libraries of size quality inspection qualification to require to blend together into upper machine sequencing sample according to sequencing, then takes 1uL
It is detected into row agarose gel electrophoresis, as shown in Figure 1.From the results, it was seen that the concentration and pillar location of institute's sample mixing sheet accord with
It closes and is expected, can be used for machine sequencing.
(3) mixed library is sequenced for MiSeq.
7.MiSeq is sequenced
After the dilution denaturation of mixed library, in loading to MiSeq reagent troughs, chip is put, after arrange parameter, start
Sequencing reaction.The data that sequencing generates carry out raw letter analysis.
7.1 data Quality Controls
The data that lower machine is obtained carry out preliminary quality screening using FastQC (version v0.11.3) software, obtain
To reads mass Data-Statistics, judge whether sequencing is qualified according to statistical result.Next cutadapt (version are used
1.8.1) the connector and primer sequence on software removal reads.Then according to data cases, filtration parameter is set, fastx is used
Tools kit carry out the filtering of low quality reads, and filtration parameter uses " 20-p 80 of-q ", i.e., if the quality of certain reads
Base number of the value less than Q20 is more than the 80% of the reads base sums, that is, filters this reads.Use iTools Fqtools
The front and back data volume of statistics filtering.
7.2 germinal mutations detect
By the reference gene group of filtered comparing the Huis.Using bwa (version0.7.12-r1039) softwares into
Row reads is compared, and alignment algorithm is " bwa mem ", and it is hg19 (GRCh37) to compare the reference gene group used.Use GATK
(version 3.3-0-g37228af) be compared data mass value correction (BaseRecalibrator modules and
PrintReads modules).The detection (HaplotypeCaller modules) of SNP and INDEL is carried out using GATK.
7.3 variation annotations and population statistics analysis
Obtained original variation file is used into ANNOVAR (Revision:
D0d72aa1e51ca9f3dc6b0611a4972956d6e93dec) software is annotated, and annotations database uses refGene,
SNP138 and thousand human genomes (2015aug).Population analysis use R (version3.1.3), statistics germline SNP and
Germline INDEL are in endometriosis group and compare the frequency occurred in health population and calculate frequency, go forward side by side
Row Fei Sheer accurately examines (Fisher ' s exact test) to calculate each germline mutation in mullerianosis
The significance level of difference in disease and normal population, and carry out the multiple testing adjustment (p-value of the FDR controls of p value
multiple test correction,FDR)。
8. using disease-susceptible humans loci gene type, frequency and disease incidence in Chinese population, according to multiplying property risk
Model analyzes disease incidence risk.Detailed process includes step 9~16.
9. crowd's genotype frequency
Frequency source in susceptibility loci Chinese population is in thousand people (1000genomes) project.The FTP provided from thousand people
Location (ftp://ftp.ncbi.nih.gov/1000genomes/ftp/release/20130502/) download idiotype number
According to choosing Chinese han population CHB, CHS totally 208 samples, calculate the ratio of each genotype as crowd's genotype frequently
Rate.
10. disease incidence
The global diseases in 2012 that disease incidence is announced from the international cancer research institution of World Health Organization subordinate
Sick epidemiological statistics data (GLOBOCAN 2012) choose the corresponding Endometriosis incidence rate data of Chinese population.
11. calculating the value-at-risk of single locus
Disease incidence is calculated as Pr (D), and 3 kinds of genotype are respectively G1, G2, G3, the incidence under the conditions of different genotype
For Pr (D | Gm), according to total probability formula
Pr (D)=Pr (D | G1) Pr (G1)+Pr (D | G2) Pr (G2)+Pr (D | G3) Pr (G3) (1)
OR1, OR2, OR3 correspond to the odds ratio (odds ratio) under 3 kinds of genotype respectively, and wherein OR1 is fixed for low-risk
Justice is that 1, OR2 is odds ratio under heterozygote genotype, and OR3 is the odds ratio under high risk, usually using patrolling in GWAS researchs
(logit-additive) pattern can be added by collecting, then OR3=OR2.It is defined according to OR
Combinatorial formula (1) calculates Pr (D | Gm), the value-at-risk OR under the genotypem
12. calculating the value-at-risk of multidigit point
Assuming that each disease-susceptible humans site, which meets, breathes out low temperature Burger that balance (HWE), independent inheritance, independent effect disease wind
Danger, the then risk under multidigit point
ORC=Π ORm (5)
13. calculating disease incidence risk
It is defined according to OR, the disease incidence under the conditions of idiotype
Compare the disease incidence that the incidence and GLOBOCAN 2012 are issued, you can know individual disease incidence risk
It is higher than, is equal to or less than normal population.
14. endometriosis Risk Forecast Method is assessed
14.1 the component of the gene detecting kit of endometriosis risk profile
(1) 2X PCR premixed liquids：PCR buffer solutions, magnesium ion, dNTP mixtures, archaeal dna polymerase and water；
The PCR amplification primer of (2) 24 SNP sites：Primer sequence SEQ ID NO：1-48,5 μM of each primer concentration；
(1) amplimer in library is built：Forward primer sequence SEQ ID NO：49, reverse primer sequences SEQ ID NO：
50-61,10 μM of each primer concentration；
(2) PCR product purifies magnetic bead.
Endometriosis Risk Forecast Method is assessed using the kit.
14.2 risk profile result
The assessment of disease risks prediction generally has clinical sample to assess and analog sample assessment, the latter in effect substantially etc.
It is same as the former, due to its economical and effective, is used in disease research.
14.2.1 the risk evaluation result of analog sample
(1) sample simulation
Using endometriosis risk loci gene type frequency and endometriosis Chinese population incidence, adopt
With bioinformatics technique, the genotype of 100000 individual of stochastic simulation；Endometriosis wind in being studied according to GWAS
Danger value OR calculates each individual disease incidence risk using formula (1)~(6)；The random number between 0-1 is simulated, if individual
Disease incidence risk is higher than random number then illness, otherwise not illness.
(2) AUC is calculated
AUC (area under Receiver operating curve ROC) is usually used in the performance of assessment models.According to analog sample gene
Type and formula (1)~(6) calculate individual disease incidence risk, and calculate AUC according to individual phenotype.The step uses R language
PredictABEL packets complete, the result is shown in Figure 1.AUC results 0.693 (0.686-0.7), can preferably show prediction effect.
14.2.2 the risk evaluation result of clinical sample
(1) clinical sample selection and operating method
100 blood cell samples are acquired, wherein being diagnosed as the blood cell sample 80 of endometriosis patients, normally
The blood cell sample of people 20.Using the gene detecting kit of endometriosis risk profile, to this 100 samples
GDNA carries out the enrichment of target site, builds library, sequencing and raw letter analysis, and step is with reference to the 1-7 in embodiment 1.
If being significantly larger than normal sample using the positive rate of kit detection mutation in illness sample, illustrate the reagent
Box can be used for the prediction of ordinary people's endometriosis risk from the point of view of Clinical results.
(2) quality of data and amplicon covering
As shown in figure 3, by taking the raw data of a sample as an example, it is observed that the length of reads is 125bp, reads
Most of base sequencing quality all 30 or more, whole sequencing quality is higher.As shown in figure 3, the reads numbers that filtering is front and back
Amount is about in 0.2~0.6M.By taking a sample as an example, by position in the target area mean coverage of filtered quality data
Numerical value is about 13656.88, maximum value 56475.37, minimum value 220.44.As shown in Figure 4, Figure 5, by filtered height
Coverage of the target area mean coverage of qualitative data much larger than the 30X needed for germinal mutation.
(3) variation detection and population analysis
By table 6 as it can be seen that in the group of 100 individuals, 19 mutation are found out altogether, wherein there are two INDEL mutation to be happened at
The intergenic region of the two genes of OR1A1 and OR1D4, the kind group frequency in the group with endometriosis
(MAF) it is far above in normal control kind group frequency (p<0.05,Fisher’s exact test,FDR multiple
Correction), while being also far above the kind group frequency that the mutation records in thousand human genomes.The sample used in clinical test
Measure it is limited in the case of, can still be found using this kit in diseased individuals there are MAF be far above normal individual several variations,
Thus illustrate, this kit has feasibility in the prediction of Chinese population endometriosis risk.
It should be noted that although the various embodiments described above have been described herein, it is not intended to limit
The scope of patent protection of the present invention.Therefore, based on the present invention innovative idea, to embodiment described herein carry out change and repair
Change, or using equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it directly or indirectly will be with
Upper technical solution is used in other related technical areas, is included within the scope of patent protection of the present invention.
Claims (7)
1. a kind of genetic test primer of endometriosis risk profile, it is characterised in that：The primer includes detection
The PCR amplification primer of 24 SNP sites of human genome.
2. the genetic test primer of endometriosis risk profile according to claim 1, it is characterised in that：It is described
Primer further include sequencing library structure amplimer.
3. the genetic test primer of endometriosis risk profile according to claim 1, it is characterised in that：It is described
24 SNP sites be rs12024204, rs801112, rs16826658, rs7521902, rs10508881,
rs11193561、rs10431397、rs3596、rs10859871、rs1268843、rs12449465、rs4141819、
rs1250248、rs13394619、rs6757804、rs1519761、rs6907340、rs7739264、rs12700667、
rs2270221、rs1333049、rs10975519、rs1537377、rs10965235。
4. the genetic test primer of endometriosis risk profile according to claim 3, it is characterised in that：
The PCR amplification primer sequence in the sites rs12024204 is respectively SEQ ID NO：1 and SEQ ID NO：25；
The PCR amplification primer sequence in the sites rs801112 is respectively SEQ ID NO：2 and SEQ ID NO：26；
The PCR amplification primer sequence in the sites rs16826658 is respectively SEQ ID NO：3 and SEQ ID NO：27；
The PCR amplification primer sequence in the sites rs7521902 is respectively SEQ ID NO：4 and SEQ ID NO：28；
The PCR amplification primer sequence in the sites rs10508881 is respectively SEQ ID NO：5 and SEQ ID NO：29；
The PCR amplification primer sequence in the sites rs11193561 is respectively SEQ ID NO：6 and SEQ ID NO：30；
The PCR amplification primer sequence in the sites rs10431397 is respectively SEQ ID NO：7 and SEQ ID NO：31；
The PCR amplification primer sequence in the sites rs3596 is respectively SEQ ID NO：8 and SEQ ID NO：32；
The PCR amplification primer sequence in the sites rs10859871 is respectively SEQ ID NO：9 and SEQ ID NO：33；
The PCR amplification primer sequence in the sites rs1268843 is respectively SEQ ID NO：10 and SEQ ID NO：34；
The PCR amplification primer sequence in the sites rs12449465 is respectively SEQ ID NO：11 and SEQ ID NO：35；
The PCR amplification primer sequence in the sites rs4141819 is respectively SEQ ID NO：12 and SEQ ID NO：36；
The PCR amplification primer sequence in the sites rs1250248 is respectively SEQ ID NO：13 and SEQ ID NO：37；
The PCR amplification primer sequence in the sites rs13394619 is respectively SEQ ID NO：14 and SEQ ID NO：38；
The PCR amplification primer sequence in the sites rs6757804 is respectively SEQ ID NO：15 and SEQ ID NO：39；
The PCR amplification primer sequence in the sites rs1519761 is respectively SEQ ID NO：16 and SEQ ID NO：40；
The PCR amplification primer sequence in the sites rs6907340 is respectively SEQ ID NO：17 and SEQ ID NO：41；
The PCR amplification primer sequence in the sites rs7739264 is respectively SEQ ID NO：18 and SEQ ID NO：42；
The PCR amplification primer sequence in the sites rs12700667 is respectively SEQ ID NO：19 and SEQ ID NO：43；
The PCR amplification primer sequence in the sites rs2270221 is respectively SEQ ID NO：20 and SEQ ID NO：44；
The PCR amplification primer sequence in the sites rs1333049 is respectively SEQ ID NO：21 and SEQ ID NO：45；
The PCR amplification primer sequence in the sites rs10975519 is respectively SEQ ID NO：22 and SEQ ID NO：46；
The PCR amplification primer sequence in the sites rs1537377 is respectively SEQ ID NO：23 and SEQ ID NO：47；
The PCR amplification primer sequence in the sites rs10965235 is respectively SEQ ID NO：24 and SEQ ID NO：48.
5. the genetic test primer of endometriosis risk profile according to claim 2, it is characterised in that：It is described
The amplimer of sequencing library structure include 1 positive universal primer and 12 reverse primers, the sequence of positive universal primer
For SEQ ID NO：49, reverse primer sequences are SEQ ID NO：50-61.
6. a kind of gene detecting kit of endometriosis risk profile, it is characterised in that：The kit includes
Primer, PCR premixed liquids and PCR product described in claim 2 purify magnetic bead.
7. the gene detecting kit of endometriosis risk profile as claimed in claim 6 is in endometriosis
Application in onset risk prediction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111334868A (en) * | 2020-03-26 | 2020-06-26 | 福州福瑞医学检验实验室有限公司 | Construction method of novel coronavirus whole genome high-throughput sequencing library and kit for library construction |
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| CN101124340A (en) * | 2005-02-18 | 2008-02-13 | 美国政府健康及人类服务部 | Identification of molecular diagnostic markers for endometriosis in blood lymphocytes |
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| CN101124340A (en) * | 2005-02-18 | 2008-02-13 | 美国政府健康及人类服务部 | Identification of molecular diagnostic markers for endometriosis in blood lymphocytes |
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| DALE R NYHOLT等: "genome-wide association meta-analysis identifies new endometriosis risk loci", 《NAT GENET》 * |
| HANS M ALBERTSEN等: "genome-wide association study link novel loci to endometriosis", 《PLOS ONE》 * |
| LUCA PAGLIARDINI等: "an Italian association study and meta-analysis with previous GWAS confirm WNT4,CDKN2BAS and FN1 as the first identified susceptibility loci for endometriosis", 《J MED GENET》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334868A (en) * | 2020-03-26 | 2020-06-26 | 福州福瑞医学检验实验室有限公司 | Construction method of novel coronavirus whole genome high-throughput sequencing library and kit for library construction |
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Application publication date: 20180828 |