CN111500723A - Marker combination for detecting premature ovarian failure genes and detection kit - Google Patents
Marker combination for detecting premature ovarian failure genes and detection kit Download PDFInfo
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Abstract
The invention discloses a marker combination and a detection kit for detecting premature ovarian failure genes, wherein the marker combination for detecting premature ovarian failure genes consists of 47 mutation sites of seventeen genes, namely BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE 1.
Description
Technical Field
The application relates to the field of premature ovarian failure detection, in particular to a marker combination and a detection kit for premature ovarian failure gene detection.
Background
In recent years, Premature Ovarian Failure (POF) gradually becomes a common disease and a frequently encountered disease in gynecologic outpatient service, and is widely concerned and emphasized in various social circles. The cause of premature ovarian failure is complex, and the treatment difficulty is great; it is clinically manifested as ovarian failure, which in turn leads to increased levels of follicle stimulating hormone and decreased levels of estrogen, ultimately leading to amenorrhea before the age of 40.
Moreover, investigation and research show that premature ovarian failure not only causes reproductive disorders such as amenorrhea and infertility, but also causes symptoms such as systemic vascular dysfunction, night sweat, depression and anxiety syndrome, and the like, and seriously affects the quality of life and physical and mental health. At present, no exact conclusion is clinically made on the pathogenesis of premature ovarian failure; clinical studies have shown that premature ovarian failure may be associated with metabolic abnormalities, autoimmune factors, genetic and external infections, and the like.
With the continuous development of molecular biology and genetics, the research on the pathogenic genes of premature ovarian failure is deepened day by day, and the premature ovarian failure is recognized to have obvious family aggregation tendency and is a complex multi-factor and polygenic genetic disease. Known POF pathogenic genes are mainly follicle development related genes, endocrine function related genes and Mendelian genetic disease related genes affecting ovarian function.
Although some genes and gene mutations related to premature ovarian failure have been found through research at home and abroad, the research on the correlation among different gene mutation sites of premature ovarian failure is less in various countries so far. The detection of single or individual genes cannot accurately and effectively reflect the premature ovarian failure condition, and a plurality of found genes and sites cannot be detected completely, so that the workload is huge, the efficiency is low, and the clinical detection and use requirements are difficult to meet. At present, no complete related research and report about the premature ovarian failure gene detection marker exists. In addition, the method finds the correlation among different gene mutation sites of premature ovarian failure, and has important scientific significance for understanding the pathogenesis of premature ovarian failure and detecting premature ovarian failure from the aspect of genetics.
Disclosure of Invention
The application aims to provide a novel marker combination and a detection kit particularly for detecting premature ovarian failure genes.
The following technical scheme is adopted in the application:
one aspect of the application discloses a marker combination for detecting premature ovarian failure genes, which consists of 47 mutation sites of seventeen genes, namely BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE 1.
The application discovers that 47 mutation sites of seventeen genes, namely BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1, can effectively realize premature ovarian failure detection, namely only 47 mutation sites of the seventeen genes of the premature ovarian failure gene detection marker combination can be detected to accurately and effectively judge the premature ovarian failure condition, so that the premature ovarian failure gene detection marker combination can be used for early diagnosis, risk assessment or treatment prognosis of premature ovarian failure.
Preferably, in the marker combination for detecting premature ovarian failure genes, 47 mutation sites and mutation information thereof are shown in table 1.
The application also discloses application of the marker combination for detecting the premature ovarian failure gene in preparing a premature ovarian failure detection reagent.
It can be understood that the gene detection marker combination can be used for detecting and judging premature ovarian failure of women, and primers or probes for detecting 47 sites of seventeen genes can be developed on the basis of the marker combination; therefore, the application creatively provides the application of the gene detection marker combination in preparing the premature ovarian failure detection reagent.
In another aspect, the present application discloses a reagent for detecting premature ovarian failure, which comprises a primer set for amplifying 47 mutation sites of seventeen genes in the marker combination for detecting premature ovarian failure genes.
Preferably, the primer set in the reagent of the present application is composed of primers having sequences shown in Seq ID No.1 to Seq ID No.94, wherein the sequences shown in Seq ID No.1 and Seq ID No.2 are primers of the first group, the sequences shown in Seq ID No.3 and Seq ID No.4 are primers of the second group, and so on, the sequences shown in Seq ID No.93 and Seq ID No.94 are primers of the forty-seventh group, and the total number of the primers is forty-seven; forty-seven sets of primers were used in sequence to amplify the 47 mutation sites as shown in Table 1 in claim 2.
It is understood that the primer sets of the sequences shown in Seq ID No.1 to Seq ID No.94 are only primers specifically used in one implementation of the present application, and do not exclude that other primers can also be used for PCR amplification of 47 mutation sites of seventeen genes. Of course, on the basis of the primer set of the present application, addition or deletion of several bases can be performed on the basis of the primer set of the present application without affecting site amplification.
In another aspect, the present application discloses a premature ovarian failure gene detection kit containing the reagent of the present application.
Preferably, the kit of the present application further comprises at least one of a DNA extraction reagent, a PCR amplification reagent, and an agarose gel electrophoresis reagent.
It can be understood that, for the convenience of use, the reagent of the present application can be combined with other existing reagents, such as a DNA extraction reagent, a PCR amplification reagent, an agarose gel electrophoresis reagent, etc., to be used as a kit; of course, the kit may further comprise other reagents for facilitating the detection of 47 mutation sites of the seventeen genes, and is not particularly limited herein.
The beneficial effect of this application lies in:
the marker combination for detecting the premature ovarian failure gene is originally established and is particularly used for detecting the premature ovarian failure gene of women. According to the detection reagent and the kit developed by the gene detection marker combination, the premature ovarian failure can be accurately, sensitively and specifically detected; in addition, the premature ovarian failure condition and risk can be more accurately and comprehensively known and judged, and an important reference basis is provided for early diagnosis, risk assessment and prognosis. The premature ovarian failure gene detection marker combination provides a new approach and scheme for clinical detection, pathogenesis and genetic research of premature ovarian failure.
Drawings
FIG. 1 is a diagram showing the results of agarose gel electrophoresis of a part of PCR amplification products in examples of the present application.
Detailed Description
The present application will be described in detail below with reference to specific embodiments and drawings. The following examples are intended to be illustrative of the present application and should not be construed as limiting the present application.
Example one
1 materials and methods
1.1 materials
1.1.1 subjects
(1) Premature ovarian failure case group: 192 premature ovarian failure patients were sampled from the special outpatient clinic of premature ovarian failure in Xinhua hospital from 2007 to 2008.
(2) Normal control group: 192 healthy volunteers from Shanghai university and university of eastern China. And (3) inclusion standard: no diseases related to ovaries, such as premature ovarian failure and ovarian cancer, exist in the third generation and the third generation of direct relatives.
The above two groups have no relationship with each other.
1.1.2 Primary reagents
1.1.2.1 DNA extraction reagent
Genomic DNA extraction A blood genomic DNA extraction kit from QIAGEN was used.
1.1.2.2 PCR reaction reagent
(1) TaKaRa Ex Taq Hot Start Version available from Takara Bio-engineering (Dalong) Ltd, containing TaKaRa Ex Taq HS (5U/. mu. L) and 10 × Ex Taq buffer (containing Mg)2+) The resulting mixture was stored at 20 ℃ in a dNTP mixture (2.5 mM each of dATP, dTTP, dGTP and dCTP).
(2) Gold-brand Taq enzyme purchased from applied biosystems of America, containing gold-brand Taq enzyme, 10 × gold-brand Taq enzyme HS buffer solution and Mg2+(50mmol/L)。
(3) Q-solution: purchased from Jitai Biotech, Inc. -20 ℃ storage.
1.1.2.3 agarose gel electrophoresis reagent
(1)10 × TBE buffer 1L buffer containing 108 g Tris base, 55 g boric acid, 0.5 × MDTA40m L (pH8.0), and storing at room temperature.
(2) Electrophoresis loading buffers 6 ×L loading Buffer and 10 ×L loading Buffer were purchased from Takara Bio engineering (Dalian) Ltd and stored at room temperature.
(3)10mg/m L ethidium bromide (final concentration: 0.5. mu.g/m L) was stored at room temperature.
(4) Marker: purchased from Tiangen Biochemical technology (Beijing) Ltd and stored at 4 ℃.
1.1.3 Main Instrument
(1) TP600 type gradient PCR instrument (TaKaRa Co., Japan)
(2) Type 4-15 large capacity centrifuge (SIGMA company, Germany)
(3) TG L-16G type desk centrifuge (Shanghai' an pavilion scientific instrument factory)
(4) FR-980 type biological electrophoresis image analysis System (Shanghai Sundari science and technology Co., Ltd.)
(5) UV-254 dark box type ultraviolet transilluminator (Beijing Ding national biotechnology, LLC)
(6) PowerBC 3002SI type numerical control electrophoresis apparatus (Shanghai Shenneng lottery biotechnology limited)
(7) DYCP-32A type agarose level electrophoresis tank (six instruments factory in Beijing City)
(8) HE-90 horizontal groove glue maker (Shanghai Tianneng science and technology Co., Ltd.)
(9) DK-8D type electric constant temperature water tank (Shanghai Jing hong test equipment Co., Ltd.)
(10) P7021TP-6 type Glanshi microwave oven (Granshi microwave oven electric appliance Co., Ltd. in Shunde district of Buddha)
(11) JT10001 type electronic balance (Shanghai Jingtian electronic instrument Co., Ltd.)
(12) Model Q L-901 vortex machine (Qinlinbel instruments manufacture Co., Ltd., Haimen city)
(13) Manually adjustable pipettes (dalong medical devices limited).
1.2 methods
1.2.1 sample Collection and DNA extraction
1) According to the principle of informed consent, peripheral blood of patients and healthy volunteers is collected, and is anticoagulated by EDTA and stored at-80 ℃ for standby.
2) DNA extraction was performed using a blood genomic DNA extraction kit. The whole process is worn with disposable gloves and masks, so that cross contamination is avoided. The disposable articles such as gun head, cyclone, tube, etc. are sterilized under the same pressure.
3) Sequentially adding 20 mu L proteinase K and 200 mu L anticoagulation blood into a centrifugal tube of 1.5m L, carrying out cyclone shaking, fully mixing uniformly, and carrying out short-time centrifugation.
4) Adding 200 μ L anhydrous ethanol, shaking thoroughly, mixing, centrifuging for a short time, transferring to a purification column, centrifuging at 13200rpm for 1min, and discarding filtrate and collection tube.
5) The rinsing solutions Buffer AW1 and Buffer AW2 were used by adding absolute ethanol to prepare working solution, placing the column in a new collection tube, adding 500. mu. L of Buffer AW1, centrifuging at 13200rpm for 1min, and discarding the filtrate.
6) The column was returned to the collection tube, 500. mu. L of Buffer AW2 was added, and the tube was centrifuged at 13200rpm for 3min, and the filtrate and collection tube were discarded.
7) The purification column was placed in a new collection tube, centrifuged at 13200rpm for 1min, and the filtrate and collection tube were discarded.
8) And (4) putting the purification column into a new centrifugal tube of 1.5m L, and standing at room temperature for 15 minutes to ensure that the residual rinsing liquid in the adsorption film is evaporated to be clean as much as possible.
9) 200 mu L of Buffer AE preheated at 68 ℃ is added into the purification column, the mixture is placed for 5 to 10min at room temperature, and the mixture is centrifuged for 2min at 13200rpm, and the obtained filtrate is the genome DNA.
The purity of all DNA samples meets the following standards that A260/A280 is between 1.7 and 2.0, A260/A230 is more than 1.5, agarose gel electrophoresis detection main band is more than 20K and has no obvious degradation, the concentration of the DNA samples is not less than 50 ng/mu L, the total DNA amount of each sample is not less than 6 mu g, and the genomic DNA is stored at the temperature of minus 20 ℃.
1.2.3 selection of mutation sites and design and Synthesis of primers
Specific amplification primers are designed for 47 mutation sites of seventeen genes, namely BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1, the information of the 47 mutation sites of the seventeen genes is shown in Table 1, the designed specific amplification primers are shown in Table 2, and the primers are synthesized by Shanghai biological engineering and technical service Limited.
TABLE 1 information on the mutation sites to be tested
TABLE 2 mutant site-specific amplification primers
In Table 2, "mutation site name" 138945734 "indicates NC-000003.12: g.138945734-138945735 insC site of FOX L2 gene," 138945951 "indicates NC-000003.12: g.138945951A > T site of FOX L2 gene, and the other site names are the same as those in Table 1. in addition," Seq ID No. "there are two sequence numbers in the order of F-terminal primer and R-terminal primer, for example," 1,2 "indicates that F-terminal primer of FOX L2 gene" 138945734 "site is Seq ID No.1, R-terminal primer is Seq ID No.2, and the rest" Seq ID No. "is so on.
1.2.4 PCR amplification
1.2.4.1 HotStart PCR
(1) HotStart PCR reaction System with a total reaction volume of 10. mu. L
(2) HotStart PCR amplification reaction conditions:
95℃5min
then 40 cycles are entered: 95 ℃ for 30s, annealing temperature for 45s and 72 ℃ for 1min
72 ℃ for 10min after the circulation is finished
Finally, stand by at 4 ℃
Here, the annealing temperature used in this example is a temperature obtained by subtracting 5 ℃ from the Tm value of each pair of primers.
1.2.5 detection of PCR products by agarose gel electrophoresis
After the PCR amplification reaction is finished, uniformly mixing a PCR product 5 mu L with a1 mu L electrophoresis loading buffer solution, then carrying out electrophoresis on 2.0% (w/V) agarose gel, adding ethidium bromide with the final concentration of 0.5 mu g/m L into the agarose gel in the preparation process, carrying out electrophoresis in 0.5 × TBE buffer solution according to the voltage of 5V/cm, carrying out gel photographing on an FR-980 type biological electrophoresis image analysis system after the electrophoresis is finished, and analyzing the PCR amplification condition.
1.2.6 PCR product sequencing detection
The method comprises the steps of sending a sample with positive agarose gel electrophoresis to a biological gene sequencing company for sequencing, carrying out marker combination modeling according to a sequencing result and real clinical information of each sample, specifically, sequencing the importance of genes and mutation sites by adopting a random forest analysis method, selecting the mutation sites of the importance top n (n is 1,2, 17) genes, and carrying out modeling analysis to determine an optimal model of the female premature ovarian failure gene detection marker combination.
2 results
2.1 agarose gel electrophoresis detection of PCR products
The agarose gel electrophoresis results of the PCR amplification products show that the specific primers of 47 sites of 17 genes can amplify target sequences which accord with expected sizes, and partial results are shown in FIG. 1. in FIG. 1, a lane M is a 100bp DNA L adapter Marker, and lanes 1 to 16 are the amplification results of the first 16 groups of primers in Table 2, namely the PCR amplification results of the primer pairs with the sequences shown in the Seq ID No.1 to 32.
2.2 Hardy-Weinberg equilibrium goodness of fit test results
Hardy-Weinberg equilibrium goodness of fit test is respectively carried out on 47 mutation sites in an ovarian premature senility case group and a normal control group, and P values are all larger than 0.05, which indicates that the 47 mutation sites are in a genetic equilibrium state in the case group and the control group and have group representativeness.
2.3 genotype and allele frequency distribution of each mutation site between case group and control group
The analysis result shows that 47 mutant sites have obvious difference in the genotypes of the premature ovarian failure case group and the normal control group, and the 47 mutant sites in the example can distinguish and detect the premature ovarian failure.
2.4 premature ovarian failure Gene detection marker combination
In this example, 192 cases of premature ovarian failure were analyzed as positive and 192 normal controls as negative, and a logistic stedt regression model was established:
x＝a0+a1*gene1+...+an*genen
wherein, a0、a1…anIs a constant; is a parameter obtained by modeling sample actual data, e is a natural constant, gene1…genenFor mutation site genes, for one sample, substituting 1 for mutation site genes and 0 for non-mutation site genes, and x of each sample can be directly calculated.
Substituting the obtained fitting value x into a sigmoid function to solve an equation, and directly modeling through R.
f(x)＝1/(1+e-x)
Obtaining a fitting result f (x), namely an ovarian premature senility risk index, and further judging whether the sample is positive or negative:
f (x) >0.5 is positive
f (x) <0.5 is negative
The results show that 17 genes of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1 in the example can accurately, comprehensively and effectively reflect the premature ovarian failure condition, and can be used as a marker combination diagnosis model.
The marker combination diagnosis model constructed in the present example was used to randomly extract samples for verification, and AUC variation trend of multiple gene stacks was analyzed, and the results are shown in table 3.
TABLE 3 Effect of different combinations of Gene mutations on marker combination models
The "number of genes" in table 3 is 1 to 17 genes selected in order of top n, i.e., BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5a1, POF1B, SGO2, STAG3, and syce1 the results of table 3 show that premature ovarian failure was diagnosed using 17 gene combinations as marker combinations with a sensitivity of 0.99 and a specificity of1, AUC 0.99.
Discussion of 3
Premature ovarian failure is a complex multi-factor, polygenic genetic disease. Multiple genes may control expression of a phenotype characteristic of premature ovarian failure, either individually or in combination, and the last or common pathway of such control is premature ovarian failure. Finding out the related genes has important scientific significance for understanding the pathogenesis of premature ovarian failure and detecting the premature ovarian failure from the genetic aspect.
3.1 Single site analysis
This example was conducted in a case-control study of a total of 384 women in 192 women including those with premature ovarian failure and healthy volunteers. The single-point analysis was performed on 47 candidate mutation sites of the above 17 genes.
The research result also shows that 47 candidate mutation sites have obvious correlation with the onset of premature ovarian failure of women, and the condition or risk of premature ovarian failure can be effectively judged only by detecting 47 mutation sites of 17 genes in the example.
3.2 higher order interaction analysis
The multi-factor dimensionality reduction method proposed by Ritchie et al in 2001 is the mainstream statistical method for researching the correlation relationship of multiple SNP sites at present. In the embodiment, the MDR method is adopted to carry out high-order interaction analysis on a plurality of mutation sites, so that a more ideal marker combination model for detecting the premature ovarian failure genes can be obtained. Because the sample size of the experiment is limited, the total number of mutation sites participating in statistical analysis cannot be excessive, otherwise the efficiency of MDR analysis is influenced.
In the experiment, seventeen genes of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1 are related to premature ovarian failure and are premature ovarian failure genes which are repeated in more than 10 samples when a single-point analysis is carried out on mutation sites, so that only 47 candidate mutation sites of 5 premature ovarian failure genes which are repeated in more than 10 samples are selected for research when a multi-point high-order interaction analysis is carried out by adopting an MDR method to preliminarily establish a premature ovarian failure gene detection marker combination model.
Finally, the analysis of the example shows that each premature ovarian failure detection index of the 47 site model is superior to that of other models, so the 47 site model is selected as the optimal gene detection marker combination model, and the marker combination model 'If-the rules' shows the premature ovarian failure classification of all genotype combinations formed by 47 mutation sites of seventeen genes of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1, including susceptible type and non-susceptible type.
In conclusion, 47 mutation sites of seventeen genes of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1 are related to the incidence of premature ovarian failure of women and are main effect sites, and the rest mutation sites or genes are non-main effect sites, so that seventeen mutation sites of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1 become the best marker combination detection model established in the research.
The establishment of the female premature ovarian failure gene detection marker combination model is the first time in China, and has innovation.
4 conclusion
The example constructs a female premature ovarian failure gene detection marker combination model consisting of 47 mutation sites of seventeen genes of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1, has good premature ovarian failure detection efficiency, and can be used for detecting or screening the premature ovarian failure of female.
Example two
Specifically, Chinese female cases collected in Xinhua hospital and 192 control cases are taken as research objects, 47 mutation sites of 17 genes are genotyped, a mainstream statistical method for researching the correlation relationship of the multiple mutation sites, namely a multi-factor dimensionality reduction Method (MDR) is adopted for statistical analysis, the female ovary gene detection marker combination model consisting of mutation sites of BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE1 is preliminarily established, the female ovary gene detection marker combination model has good early detection efficiency, the early detection marker combination model has early diagnosis sensitivity of 0% OR 96, the early diagnosis marker combination model has low early diagnosis significance, the ovarian failure diagnosis marker combination model has low early diagnosis sensitivity, the early diagnosis marker combination model has early diagnosis significance of ovary failure, the early diagnosis marker combination model has early diagnosis significance of 100% OR 96, the early diagnosis marker combination model has early diagnosis marker classification sensitivity of early premature aging, the ovarian failure diagnosis marker combination model has early diagnosis marker classification significance of 100.96%, the early diagnosis marker classification of the female ovary diagnosis marker is established by adopting the following specific genetic combination model:
1. aiming at the constructed female premature ovarian failure gene detection marker combination model, a detection kit of 47 sites of seventeen genes is developed. The kit consists of 47 boxes, and each small box corresponds to one locus in a 47-locus model for genotyping. Each small box is filled with relevant reagents for DNA extraction, PCR amplification and agarose gel electrophoresis.
2. The detection kit is suitable for potential premature ovarian failure patients scattered in a community, namely those with personal or family allergy history but no premature ovarian failure, and the potential patients can develop premature ovarian failure under certain conditions. The concept of three-level prevention of premature ovarian failure is proposed abroad, wherein the first-level prevention is to take active measures when the disease does not occur, such as changing the living environment, dietary habits and the like of high risk groups, and preventing the premature ovarian failure. The female premature ovarian failure gene detection kit can be used for early screening potential patients to determine the high risk group of premature ovarian failure, and measures such as allergen avoidance are taken in a targeted manner to prevent the premature ovarian failure, so that the prevalence rate of premature ovarian failure is effectively reduced, and a large amount of resources are saved.
3. A detection step: (1) collecting peripheral blood of a subject; (2) extracting peripheral blood genome DNA; (3) carrying out PCR amplification on the genome DNA by adopting specific primers of 47 sites; (4) detecting the PCR product by agarose gel electrophoresis; (5) performing high-throughput sequencing analysis on the PCR product; (6) obtaining the genotypes of 47 mutation sites; (7) judging the premature ovarian failure condition and risk according to the genotype result of the 47 sites, namely determining whether the tested person has premature ovarian failure or whether the tested person is at high risk, low risk or not difficult to feel; (8) and performing premature ovarian failure education and management on premature ovarian failure high-risk persons and families thereof.
The foregoing is a detailed description of the present application in connection with specific embodiments thereof, and implementations of the present application are not to be considered limited to those descriptions. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the basic inventive concepts herein.
Sequence listing
<110> Guangzhou Dakang Gene technology Co., Ltd
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<400>89
caatggagaa cattgcagcc 20
<210>90
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>90
gaaagaggaa attccatgtc 20
<210>91
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>91
tatgcccata tcacacaatt c 21
<210>92
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>92
gtgaatttgt ccaaagcagg ta 22
<210>93
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>93
ggcagattcc atagccatac 20
<210>94
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>94
acagacggca agaggtaatg 20
Claims (7)
1. A marker combination for detecting premature ovarian failure genes is characterized by consisting of 47 mutation sites of seventeen genes, namely BMP15, EIF2B2, ERCC6, ESR1, FIG L A, FOX L2, FSHR, HFM1, MCM8, MCM9, MSH5, NOBOX, NR5A1, POF1B, SGO2, STAG3 and SYCE 1.
2. A marker combination according to claim 1, wherein: the 47 mutation sites and the mutation information thereof are shown in Table 1,
TABLE 1
3. Use of the marker combination for premature ovarian failure gene detection according to claim 1 or 2 in preparation of a premature ovarian failure detection reagent.
4. An agent for detecting premature ovarian failure, which is characterized in that: the reagent comprises a primer group for amplifying 47 mutation sites of seventeen genes in the marker combination for detecting the premature ovarian failure genes as claimed in claim 2.
5. The reagent according to claim 4, characterized in that: the primer group consists of primers of sequences shown by Seq ID No.1 to Seq ID No.94, wherein the sequences shown by Seq ID No.1 and Seq ID No.2 are a first group of primers, the sequences shown by Seq ID No.3 and Seq ID No.4 are a second group of primers, and so on, the sequences shown by Seq ID No.93 and Seq ID No.94 are forty-seventh groups of primers, and the total number of the primers is forty-seven; forty-seven sets of primers were used in sequence to amplify the 47 mutation sites as shown in Table 1 in claim 2.
6. An ovarian premature aging gene detection kit containing the reagent according to claim 4 or 5.
7. The premature ovarian failure gene detection kit according to claim 6, wherein: the kit also contains at least one of a DNA extraction reagent, a PCR amplification reagent and an agarose gel electrophoresis reagent.
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