CN111549137B - Genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof - Google Patents
Genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses a genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof, wherein the genetic molecular marker comprises 42 genetic loci. The genetic risk prediction model is constructed, and genetic risk assessment of gastric cancer is carried out on target people. Provides the specific primer of the SNP marker and the application thereof in preparing a gastric cancer auxiliary diagnosis kit. Provides a gastric cancer auxiliary diagnosis kit. The genetic marker kit for assisting in diagnosis of gastric cancer is expected to lay a foundation for screening, individuation prevention and treatment of gastric cancer high risk groups in China, and provides decision basis. The SNP genetic marker is different from the traditional biomarker, has good stability and is easy to detect. The gastric cancer auxiliary diagnosis kit is beneficial to a clinician to quickly master the genetic risk of gastric cancer of a patient, performs early diagnosis, personalized prevention and treatment, and provides decision basis for screening and intervention of gastric cancer high-risk groups.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof.
Background
At present, gastric cancer is a common malignant tumor, the incidence rate of which occupies the sixth place of all tumors in the world, and has obvious regionalism, and especially has higher incidence rate in eastern Asia areas such as China, japan, korea and the like. According to statistics, about 429 ten thousand new cancers occur in China in 2015, and 281 ten thousand patients die from the cancer, wherein 68 ten thousand stomach cancers occur, 50 ten thousand die, and all tumors are located at the second place, so that the health of human beings is seriously endangered. In recent years, along with economic development and social progress of China, the incidence rate and death rate of gastric cancer tend to be reduced year by year, but the early detection of gastric cancer is difficult, and early, specific and sensitive screening indexes are not yet available, so that the disease burden caused by the early detection of gastric cancer is still quite large. Therefore, effective markers are searched for evaluating the incidence risk of the gastric cancer, and the high-risk group is assisted with methods such as conventional gastroscopy and the like so as to realize early discovery and early diagnosis of the gastric cancer, thereby greatly reducing the incidence rate of the gastric cancer.
The occurrence and development of gastric cancer are biological processes involving multiple stages and factors, and are the result of the combination of environmental factors (such as smoking, eating habits), genetic factors and interactions between the two. However, only a few people develop gastric cancer under the same environmental exposure, suggesting that genetic factors play a key role in the occurrence and development of gastric cancer. Further, the genetic explanation degree of gastric cancer occurrence in the Chinese population is 20% found by the scholars, and the effect of genetic factors is proved. Therefore, the identification of the genetic variation site related to gastric cancer is helpful for better evaluating genetic risk of individuals for gastric cancer, so that early detection, diagnosis and targeted treatment of gastric cancer are realized.
The genome-wide association study (GWAS), a powerful means of genetic epidemiological research, is currently widely used in association studies of a variety of complex diseases. Currently, GWAS has found tens of single nucleotide polymorphism (single nucleotide polymorphism, SNP) sites associated with gastric cancer, providing a solid early foundation for genetic studies of gastric cancer. A plurality of researches show that the establishment of a gastric cancer incidence genetic risk prediction model based on the genetic locus found by the GWAS has very important significance for screening high-risk groups and realizing early prevention.
However, the conventional gastric cancer genetic model is mostly constructed by adopting a GWAS site, the prediction efficiency is low, and the effect of the genetic prediction model cannot be exerted to the greatest extent. Therefore, screening from the whole genome, combining with external data verification, developing gastric cancer genetic risk model research, identifying reliable gastric cancer auxiliary diagnosis genetic molecular markers, constructing an optimized gastric cancer onset risk prediction model, and being particularly urgent and important for more effective individuation prevention, scientific intervention and the like in future.
Through the above analysis, the problems and defects existing in the prior art are as follows: the traditional gastric cancer genetic model is mostly constructed by adopting a GWAS locus, has low prediction efficiency, and cannot exert the effect of the genetic prediction model to the greatest extent.
The difficulty of solving the problems and the defects is as follows:
how to construct a gastric cancer genetic model with better prediction efficiency from the whole gene perspective and verify the gastric cancer genetic model in external data is a challenging task.
The meaning of solving the problems and the defects is as follows:
the prediction accuracy of the gastric cancer genetic model can be effectively improved, so that the method is better used for screening high-risk groups suffering from gastric cancer.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof.
The invention is realized in such a way that a genetic molecular marker related to gastric cancer auxiliary diagnosis, genetic molecular markers related to gastric cancer auxiliary diagnosis are rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727 rs9341727, rs9341727 rs9341727, a combination of rs9341727 and rs 9341727.
The invention also aims to provide a specific amplification primer of the genetic molecular marker related to gastric cancer auxiliary diagnosis, wherein the specific amplification primer has a sequence of SEQ ID NO. 1-SEQ ID NO.166.
Another object of the present invention is to provide a specific probe primer of the genetic molecular marker related to gastric cancer auxiliary diagnosis, wherein the specific probe primer is: the primer sequence of rs4460629 is SEQ ID NO. 3-SEQ ID NO.168.
The invention also aims to provide an application of the genetic molecular marker related to gastric cancer auxiliary diagnosis in preparing a gastric cancer auxiliary diagnosis kit.
The invention also aims to provide an application of the genetic molecular marker related to gastric cancer auxiliary diagnosis in preparing a gastric cancer auxiliary diagnosis kit by using a specific amplification primer.
The invention also aims to provide an application of the genetic molecular marker related to gastric cancer auxiliary diagnosis in preparing a gastric cancer auxiliary diagnosis kit by using a specific probe primer.
Another object of the invention is to provide a gastric cancer auxiliary diagnostic kit comprising the genetic molecular marker related to gastric cancer auxiliary diagnosis, which is used for detecting the gene types of rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs3857490, rs6959127, rs4305854, rs1052969, rs9791835, rs10952328, rs7782998, rs656248, rs378363, rs2460554, rs4751153, rs9329332, rs10777647, rs35326083, rs17027488, rs319284, rs2225046, rs10483947, rs7168752, rs875339, rs3093315, rs918655, rs348793, rs17000838, 1034420 and rs 2019061.
Further, specific amplification primers and specific probe primers are contained.
Further, the kit also includes PCR reagents.
Further, the PCR reagent is Taq enzyme, dNTP mixed solution, deionized water and standard and reference substances
By combining all the technical schemes, the invention has the advantages and positive effects that: the genetic risk prediction model is constructed, and genetic risk assessment of gastric cancer is carried out on target people. Provides the specific primer of the SNP marker and the application thereof in preparing a gastric cancer auxiliary diagnosis kit. Provides a gastric cancer auxiliary diagnosis kit.
Based on the research design of Chinese crowd gastric cancer case contrast, the invention utilizes gastric cancer whole genome association research (GWAS) data and adopts a strategy of combining a minimum absolute contraction and selection operator (LASSO) and a genetic algorithm to screen a group of SNPs sites closely related to the risk of gastric cancer onset from the whole genome; on the basis, constructing a genetic risk prediction model, and evaluating the efficacy of predicting the stomach cancer incidence risk by SNPs sites in external data; finally preparing the gastric cancer auxiliary diagnosis kit, and providing technical support for early diagnosis and prevention of gastric cancer.
The genetic marker kit for assisting in diagnosis of gastric cancer is expected to lay a foundation for screening, individuation prevention and treatment of gastric cancer high risk groups in China, and provides decision basis. The SNP genetic marker is different from the traditional biomarker, has good stability and is easy to detect. The gastric cancer auxiliary diagnosis kit is beneficial to a clinician to quickly master the genetic risk of gastric cancer of a patient, performs early diagnosis, personalized prevention and treatment, and provides decision basis for screening and intervention of gastric cancer high-risk groups.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of gastric cancer susceptibility genetic loci screened based on LASSO algorithm provided by the embodiment of the application;
in the figure: a: LASSO coefficients of different Lambda; b: SNPs frequency figures obtained based on LASSO models of different Lambda; c: areas under ROC curves of different gastric cancer GRS models constructed based on SNPs frequency; AUC: area under ROC curve.
FIG. 2 is a schematic diagram of gastric cancer susceptibility genetic loci screened based on genetic algorithm provided by the embodiment of the application;
in the figure: a: obtaining SNPs frequency charts of the optimal 50 sets based on a genetic algorithm; b: areas under ROC curves of different gastric cancer GRS models constructed based on SNPs frequency; AUC: area under ROC curve.
FIG. 3 is an evaluation schematic diagram of a gastric cancer genetic risk prediction model provided by an embodiment of the present application;
In the figure: a: a calibration curve; b: ROC curve; AUC: area under ROC curve.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The invention provides a group of SNP genetic markers related to prediction of gastric cancer incidence risk, which are combinations of rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs3857490, rs6959127, rs4305854, rs1052969, rs9791835, rs10952328, rs7782998, rs656248, rs378363, rs2460554, rs4751153, rs9329332, rs10777647, rs35326083, rs17027488, rs319284, rs2225046, rs10483947, rs7168752, rs875339, rs3093315, rs918655, rs348793, rs17000838, 1034420 and rs 2019061.
The specific amplification primer of the SNP genetic marker comprises the following components: primer sequences of rs4460629 are SEQ ID NO.1 and SEQ ID NO.2; primer sequences of rs4451581 are SEQ ID NO.5 and SEQ ID NO.6; primer sequences of rs11802956 are SEQ ID NO.9 and SEQ ID NO.10; primer sequences of rs236322 are SEQ ID NO.13 and SEQ ID NO.14; primer sequences of rs11890919 are SEQ ID NO.17 and SEQ ID NO.18; primer sequences of rs3771790 are SEQ ID NO.21 and SEQ ID NO.22; primer sequences of rs7425197 are SEQ ID NO.25 and SEQ ID NO.26; primer sequences of rs1403651 are SEQ ID NO.29 and SEQ ID NO.30; primer sequences of rs4685983 are SEQ ID NO.33 and SEQ ID NO.34; primer sequences of rs1504359 are SEQ ID NO.37 and SEQ ID NO.38; primer sequences of rs2035057 are SEQ ID NO.41 and SEQ ID NO.42; primer sequences of rs1870563 are SEQ ID NO.45 and SEQ ID NO.46; primer sequences of rs1697952 are SEQ ID NO.49 and SEQ ID NO.50; primer sequences of rs1564601 are SEQ ID NO.53 and SEQ ID NO.54; the primer sequences of rs13195186 are SEQ ID NO.57 and SEQ ID NO.58; primer sequences of rs9341727 are SEQ ID NO.61 and SEQ ID NO.62; primer sequences of rs3857490 are SEQ ID NO.65 and SEQ ID NO.66; primer sequences of rs6959127 are SEQ ID NO.69 and SEQ ID NO.70; primer sequences of rs4305854 are SEQ ID NO.73 and SEQ ID NO.74; primer sequences of rs1052969 are SEQ ID NO.77 and SEQ ID NO.78; primer sequences of rs9791835 are SEQ ID NO.81 and SEQ ID NO.82; primer sequences of rs10952328 are SEQ ID NO.85 and SEQ ID NO.86; primer sequences of rs7782998 are SEQ ID NO.89 and SEQ ID NO.90; primer sequences of rs656248 are SEQ ID NO.93 and SEQ ID NO.94; primer sequences of rs378363 are SEQ ID NO.97 and SEQ ID NO.98; primer sequences of rs2460554 are SEQ ID NO.101 and SEQ ID NO.102; primer sequences of rs4751153 are SEQ ID NO.105 and SEQ ID NO.106; primer sequences of rs9329332 are SEQ ID NO.109 and SEQ ID NO.110; primer sequences of rs10777647 are SEQ ID NO.113 and SEQ ID NO.114; primer sequences of rs35326083 are SEQ ID NO.117 and SEQ ID NO.118; primer sequences of rs17027488 are SEQ ID NO.121 and SEQ ID NO.122; primer sequences of rs319284 are SEQ ID NO.125 and SEQ ID NO.126; primer sequences of rs2225046 are SEQ ID NO.129 and SEQ ID NO.130; primer sequences of rs10483947 are SEQ ID NO.133 and SEQ ID NO.134; primer sequences of rs7168752 are SEQ ID NO.137 and SEQ ID NO.138; primer sequences of rs875339 are SEQ ID NO.141 and SEQ ID NO.142; primer sequences of rs3093315 are SEQ ID NO.145 and SEQ ID NO.146; primer sequences of rs918655 are SEQ ID NO.149 and SEQ ID NO.150; primer sequences of rs348793 are SEQ ID NO.153 and SEQ ID NO.154; primer sequences of rs17000838 are SEQ ID NO.157 and SEQ ID NO.158; primer sequences of rs1034420 are SEQ ID NO.161 and SEQ ID NO.162; primer sequences of rs2019061 are SEQ ID NO.165 and SEQ ID NO.166
The specific probe primer of the SNP genetic marker comprises the following components: primer sequences of rs4460629 are SEQ ID NO.3 and SEQ ID NO.4; primer sequences of rs4451581 are SEQ ID NO.7 and SEQ ID NO.8; primer sequences of rs11802956 are SEQ ID NO.11 and SEQ ID NO.12; primer sequences of rs236322 are SEQ ID NO.15 and SEQ ID NO.16; primer sequences of rs11890919 are SEQ ID NO.19 and SEQ ID NO.20; primer sequences of rs3771790 are SEQ ID NO.23 and SEQ ID NO.24; primer sequences of rs7425197 are SEQ ID NO.27 and SEQ ID NO.28; primer sequences of rs1403651 are SEQ ID NO.31 and SEQ ID NO.32; primer sequences of rs4685983 are SEQ ID NO.35 and SEQ ID NO.36; primer sequences of rs1504359 are SEQ ID NO.39 and SEQ ID NO.40; primer sequences of rs2035057 are SEQ ID NO.43 and SEQ ID NO.44; primer sequences of rs1870563 are SEQ ID NO.47 and SEQ ID NO.48; primer sequences of rs1697952 are SEQ ID NO.51 and SEQ ID NO.52; primer sequences of rs1564601 are SEQ ID NO.55 and SEQ ID NO.56; primer sequences of rs13195186 are SEQ ID NO.59 and SEQ ID NO.60; primer sequences of rs9341727 are SEQ ID NO.63 and SEQ ID NO.64; primer sequences of rs3857490 are SEQ ID NO.67 and SEQ ID NO.68; primer sequences of rs6959127 are SEQ ID NO.71 and SEQ ID NO.72; primer sequences of rs4305854 are SEQ ID NO.75 and SEQ ID NO.76; primer sequences of rs1052969 are SEQ ID NO.79 and SEQ ID NO.80; primer sequences of rs9791835 are SEQ ID NO.83 and SEQ ID NO.84; primer sequences of rs10952328 are SEQ ID NO.87 and SEQ ID NO.88; primer sequences of rs7782998 are SEQ ID NO.91 and SEQ ID NO.92; primer sequences of rs656248 are SEQ ID NO.95 and SEQ ID NO.96; primer sequences of rs378363 are SEQ ID NO.99 and SEQ ID NO.100; primer sequences of rs2460554 are SEQ ID NO.103 and SEQ ID NO.104; primer sequences of rs4751153 are SEQ ID NO.107 and SEQ ID NO.108; primer sequences of rs9329332 are SEQ ID NO.111 and SEQ ID NO.112; primer sequences of rs10777647 are SEQ ID NO.115 and SEQ ID NO.116; primer sequences of rs35326083 are SEQ ID NO.119 and SEQ ID NO.120; primer sequences of rs17027488 are SEQ ID NO.123 and SEQ ID NO.124; primer sequences of rs319284 are SEQ ID NO.127 and SEQ ID NO.128; primer sequences of rs2225046 are SEQ ID NO.131 and SEQ ID NO.132; primer sequences of rs10483947 are SEQ ID NO.135 and SEQ ID NO.136; primer sequences of rs7168752 are SEQ ID NO.139 and SEQ ID NO.140; primer sequences of rs875339 are SEQ ID NO.143 and SEQ ID NO.144; primer sequences of rs3093315 are SEQ ID NO.147 and SEQ ID NO.148; primer sequences of rs918655 are SEQ ID NO.151 and SEQ ID NO.152; primer sequences of rs348793 are SEQ ID NO.155 and SEQ ID NO.156; primer sequences of rs17000838 are SEQ ID NO.159 and SEQ ID NO.160; primer sequences of rs1034420 are SEQ ID NO.163 and SEQ ID NO.164; primer sequences of rs2019061 are SEQ ID NO.167 and SEQ ID NO.168
The SNP genetic marker is applied to construction of a genetic risk prediction model and preparation of a gastric cancer auxiliary diagnosis kit.
The invention relates to application of a specific amplification primer of a SNP genetic marker in preparing a gastric cancer auxiliary diagnosis kit.
The invention discloses application of a specific probe primer of an SNP genetic marker in preparing a gastric cancer auxiliary diagnosis kit.
The invention provides a gastric cancer auxiliary diagnosis kit, the kit is used for detecting rs, rs in peripheral blood DNA rs, rs rs, rs rs, rs.
The gastric cancer auxiliary diagnosis kit contains the specific amplification and probe primers of the SNP genetic marker; the PCR reagent also comprises reagents commonly used in PCR, such as Taq enzyme, dNTP mixed solution, deionized water and the like; standard and control.
(1) Sample collection: determining sample inclusion criteria and exclusion criteria, the system collecting blood samples meeting the criteria;
(2) Genotype detection and site screening: selecting gastric cancer cases and healthy controls thereof, acquiring individual genotype data by using a full genome high-density SNP chip, and identifying SNPs related to the risk of gastric cancer onset by using a strategy combining LASSO and a genetic algorithm;
(3) Model evaluation: constructing a genetic risk prediction model based on SNPs with risk effect, and judging the calibration degree and the distinction degree of the model in an external data set by using a calibration curve and a subject work characteristic curve (ROC) so as to detect the prediction capability of risk SNPs sites on stomach cancer incidence risk;
(4) Preparation of gastric cancer auxiliary diagnosis kit: and constructing an auxiliary diagnosis kit based on the screened SNP genetic markers.
The technical effects of the present invention will be described in detail with reference to experiments.
The experimental method comprises the following steps:
1. collecting study objects
(1) Pathological diagnosis of gastric cancer cases;
(2) Healthy controls;
the invention incorporates 1835 gastric cancer cases and 2613 healthy controls (training set: 1625 cases and 2100 controls; test set: 210 cases and 513 controls) to identify risk sites.
2. Extracting peripheral blood genome DNA by phenol-chloroform method, and operating according to conventional method. Generally, 20-50 ng/. Mu.l of DNA can be obtained with a purity (ratio of UV 260OD to 280 OD) of 1.6-2.0.
3. Training set genome-wide SNPs site detection and risk site primary screening
(1) Taking a whole genome DNA sample of a subject in the training set, wherein the whole genome DNA sample comprises 1625 cases and 2100 controls;
(2) Genotyping was performed using Illumina Human 660W-Quad Chips genotyping Chips;
(3) Genotype filling is carried out on the training set data by adopting IMUTE 2 software;
(4) Comparing the distribution difference of each genotype in the gastric cancer cases and the control of the training set by fitting logistic regression analysis, and screening inclusion sites by taking the correlation P value <0.01 as a standard;
(5) And (3) primarily screening sites related to the incidence risk of gastric cancer by adopting a LASSO algorithm strategy.
4. TaqMan MGB probe method genotyping and risk site determination for testing single SNP in set
(1) Taking DNA samples in a test set, wherein the DNA samples comprise 210 cases and 513 controls;
(2) Designing a specific amplification primer and a probe primer of a single SNP;
(3) PCR amplification reaction;
(4) Based on the initially screened sites in the training set, genetic algorithm is further used to determine sites related to the risk of gastric cancer onset.
5. Construction and evaluation of genetic risk prediction model
(1) SNPs sites closely related to the risk of gastric cancer onset, which are identified based on the above strategy, comprise rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs3857490, rs6959127, rs4305854, rs1052969, rs9791835, rs10952328, rs7782998, rs656248, rs378363, rs2460554, rs4751153, rs9329332, rs10777647, rs35326083, rs17027488, rs319284, rs2225046, rs10483947, rs7168752, rs875339, rs3093315, rs918655, rs348793, rs17000838, rs1034420 and rs2019061;
(2) And constructing a weighted genetic risk score model (wGRS model) by taking the LASSO regression coefficient as a weight.
(3) And performing risk assessment on the test set sample by utilizing a wGRS model, and evaluating the calibration degree and the distinguishing degree of the model by utilizing a calibration curve and a ROC curve so as to detect the prediction capability of the identified risk SNPs on the stomach cancer incidence risk.
6. Preparation of gastric cancer auxiliary diagnosis kit
The SNPs (rs 4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs 7425197) rs9341727, rs9341727 rs9341727, rs 9341727. The diagnostic reagent comprises specific amplification and probe primers of the SNP genetic marker; the PCR reagent also comprises reagents commonly used in PCR, such as Taq enzyme, dNTP mixed solution, deionized water and the like; standard and control.
7. Statistical analysis
The correlation intensity of SNPs and the risk of gastric cancer onset is analyzed by adopting a logistic regression addition model, and confounding variables (such as gender and age) are adjusted. The strategy of LASSO combined genetic algorithm is adopted to screen SNPs sites related to the risk of gastric cancer incidence from the whole genome.
The genetic risk prediction model is fitted by calculating wGRS. The basic steps include:
(1) Quantitative scoring is performed on the three genotypes of each SNP, for example, the wild homozygote is "0", the heterozygote is "1", the homozygote mutant is "2", and the effect scores of all loci are adjusted to be in forward association;
(2) The weight coefficient of each SNP is the regression coefficient obtained from the LASSO regression model, and the construction equation is as follows: wgrs=rs 4460629 ×0.395+rs4451581×0.188+rs11802956×0.049+rs236322×0.102+rs11890919× 0.081+rs3771790×0.039+rs7425197×0.113+rs1403651×0.11+rs4685983×0.096+rs1504359×0.09+rs2035057 x 0.156+rs1870563 x 0.111+rs1697952 x 0.157+rs1564601 x 0.058+rs1319516x 0.073+rs9341727 x 0.123+rs3857490 x 0.167+rs6959127 x 0.114+rs4305854 x 0.112+rs1052969 x 0.052+rs9791835 x 0.085+rs10952328×0.113+rs7782998×0.253+rs656248×0.089+rs378363×0.141+rs2460554×0.05+rs4751153 x 0.093+rs9329332 x 0.096+rs10777647 x 0.123+rs35326083 x 0.016+rs17027488 x 0.065+rs319284 x 0.177+rs2225046 x 0.019+rs10483947 x 0.127+rs7168752 x 0.146+rs875339 x 0.133+rs3093315 x 0.065+rs918655 x 0.035+rs348793 x 0.095+rs170008380.073+rs1034320 x 0.088+rs2019061 x 0.117;
(3) The predicted performance of the GRS model was assessed using the calibration curve and the area under the ROC curve (AUC).
All statistical analyses were performed using PLINK1.90 and R3.5.1 software. The statistical significance P value was 0.05 and both were double-sided.
The results illustrate:
(1) The importance scores obtained based on LASSO regression are continuously fitted with the GRS model, and the cross-validated AUC is used as an evaluation index, and 1076 stomach cancer genetic loci are finally screened in a training set, wherein the AUC reaches 1 (see figure 1).
(2) Adopting genetic algorithm, taking AUC of test set as index, obtaining optimal 50 variable sets, fitting GRS model based on frequency of 50 variable sets, finally finding out that AUC of 42 genetic loci constructed GRS model in test set reaches highest (see figure 2), comprises rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057 rs1870563, rs1697952, rs1564601, rs13195186, rs9341727 rs1870563, rs1697952, rs1564601, rs13195186, rs9341727 rs9341727, rs 9341727.
(3) GRS is constructed based on the 42 stomach cancer genetic loci, and a stomach cancer onset risk prediction model is constructed through a logistic regression method. Based on the calibration curve and ROC curve, the model was found to have a good degree of calibration (goodness of fit test P-value=0.866) and degree of discrimination (AUC Training set ＝0.672；AUC Test set =0.7, see fig. 3).
Based on the above results, the present inventors prepared a gastric cancer auxiliary diagnostic kit comprising specific primers and other reagents for 42 SNPs identified.
In general, the gastric cancer auxiliary diagnosis kit prepared by the specific primers of the 42 SNPs is beneficial to a clinician to quickly grasp the genetic risk of the patient for disease, and provides powerful support for timely taking therapeutic measures.
The technical scheme of the invention is further described below with reference to specific embodiments.
Example 1 sample collection and data consolidation
Training set: the inventor obtains stomach cancer GWAS data of Chinese people from a genotype and phenotype database (dbGaP, phs000361.v1.p1), wherein the data are from a shanxi upper gastrointestinal tumor genetic project and a Lin county nutrition intervention experimental project, and comprise 1625 cases and 2100 healthy controls.
Test set: the inventor collects and sorts gastric cancer blood samples from hospitals in south Beijing, shanghai, xuzhou and other places from 2010 to 2018, wherein the gastric cancer blood samples comprise 210 gastric cancer samples and 513 comparison cases of health examination in hospitals at the same time, and collects related epidemiological and clinical data.
EXAMPLE 2 training of Whole genome scanning of SNPs in peripheral blood DNA
Genotyping was performed using an Illumina Human 660W-Quad Chips in 1625 gastric cancer cases and 2100 healthy controls meeting the requirements. The method comprises the following specific steps:
(1) Preparing a lysate: 40 portions, 219.72g sucrose, 2.02g magnesium chloride, 20ml triton X-100 (amresco 0694) were mixed and the volume was set to 2000ml using TrisHcl solution. (2) The lysate was added to the peripheral blood in a 2ml cryopreservation tube and mixed upside down. (3) removal of erythrocytes: the 5ml centrifuge tube was filled to a 4ml scale with lysate, mixed well, centrifuged at 4000rpm for 10 minutes and the supernatant was discarded. And 4ml of lysate was added to the pellet, again mixed, washed once, centrifuged at 4000rpm for 10 minutes, and the supernatant was discarded. (4) preparing an extract: each 300ml contains 122.5ml of 0.2M sodium chloride, 14.4ml of 0.5M ethylenediamine tetraacetic acid, 15ml of 10% sodium dodecyl sulfate and 148.1ml of double distilled water. (5) DNA extraction: to the obtained precipitate, 1ml of the extract and 8. Mu.l of proteinase K were added, and the mixture was thoroughly mixed by shaking on a shaker, and water-bath was carried out at 37℃overnight. (6) protein removal: 1ml of saturated phenol was added and thoroughly mixed, centrifuged at 4000rpm for 10 minutes, and the supernatant was transferred to a new 5ml centrifuge tube. An equal volume of a mixture of chloroform and isoamyl alcohol (chloroform: isoamyl alcohol=24:1, v/v) was added to the supernatant, after thorough mixing, the supernatant was centrifuged at 4000rpm for 10 minutes, and the supernatant was taken and placed into two 1.5ml centrifuge tubes, respectively. (7) DNA precipitation: 60 μl of 3M sodium acetate was added to the supernatant, and ice absolute ethanol was added in an equal volume to the supernatant, and the supernatant was gently shaken up and down to give a white flocculent precipitate, which was centrifuged at 12000rpm for 10min. (8) DNA washing: 1ml of ice absolute ethanol was added to the precipitate, the mixture was centrifuged at 12000rpm for 10 minutes, and the supernatant was discarded. And finally, placing the mixture in a clean and dry environment for drying by evaporation. (9) measuring the concentration: generally, 20-50 ng/. Mu.l of DNA can be obtained with a purity (ratio of UV 260OD to 280 OD) of 1.6-2.0. (10) whole genome scan: full genome scanning was performed on a Human 660W-Quad Chips chip. (11) genotype filling by using IMUTE 2 software; (12) data analysis and processing: screening SNPs with significant difference from a training set case group and a healthy control group by fitting a logistic regression model, and screening inclusion sites by taking a correlation P value <0.01 as a standard; further adopts a LASSO algorithm strategy to preliminarily screen the related sites of the stomach cancer incidence risk.
Example 3 TaqMan Probe genotyping for testing Single SNPs in a set
Corresponding tests were performed in 210 cases and 513 controls meeting the requirements.
(1) The DNA extraction method is the same as above. (2) TaqMan probe real-time PCR method genotyping:
(1) the probes and the primers are designed aiming at the sites which are preliminarily screened in the training set, all meet the requirements of the probes and the primers, and the typing can be successfully performed. (2) The primer was diluted to a 10-fold working concentration and the probe was diluted to a 20-fold working concentration. (3) Preparing a real-time PCR reaction system: each 5. Mu.l portion was used, which included 1.25. Mu.l of sterile double distilled water, 2.5. Mu.l of 1 XqPCR Mix (THUNDERBIRD Probe qPCR Mix, available from TOYOBD Co.), 0.25. Mu.l of each of the upstream and downstream primers (1 pmol/. Mu.l), 0.125. Mu.l of FAM probe (0.25 pmol/. Mu.l), 0.125. Mu.l of HEX probe (0.25 pmol/. Mu.l), 0.125. Mu.l of 1.25 XROX solution (0.25 pmol/. Mu.l) and 0.5. Mu.l of DNA template (10 ng/. Mu.l). (4) The prepared real-time PCR reaction system was added to a 384-well plate (available from AXYGEN, U.S.A.) by a gun (available from Eppendorf, germany) and subjected to a sealing treatment. (5) Typing of SNPs: the 384 well plate with the membrane sealed is placed in an ABI PRISM 7900HT type fluorescence quantitative PCR instrument for typing, and the experimental procedure is as follows: 1) Activating enzyme at 95deg.C for 2 min; 2) Denaturation of DNA at 95℃for 15 s; 3) The primer and probe were annealed and extended at 60℃for 60s for 40 cycles. (6) Genotyping was performed using SDS 2.4 software. Wherein blue and red represent two different homozygotes, green represents heterozygote, gray represents negative control (NTC) and x represents typing failure, respectively. (7) Data analysis: based on the initially screened sites in the training set, genetic algorithm is further used to determine sites related to the risk of gastric cancer onset.
Example 4 genetic risk prediction model was constructed using wGRS, a wGRS model was constructed using LASSO regression coefficients as weights based on the 42 risk SNPs sites identified above, and the prediction ability of risk sites against gastric cancer occurrence risk was evaluated using calibration curves and ROC curves. The results show that the identified 42 SNPs (rs 4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs3857490, rs6959127, rs4305854, rs1052969, rs9791835, rs10952328, rs7782998, rs656248, rs378363, rs2460554, rs4751153, rs9329332, rs10777647, rs35326083, rs17027488, rs319284, rs2225046, rs10483947, rs7168752, rs875339, rs3093315, rs918655, rs348793, rs17000838, rs1034420 and rs 2019061) are better able to distinguish gastric cancer cases from controls (see table 1), and that the GRS model has better degree of alignment (goodness-of-fit P value=0.866) and degree of difference (AUC) Training set ＝0.672；AUC Test set =0.7, see fig. 3), suggesting that these 42 genetic loci are better able to predict the genetic risk of gastric cancer occurrence.
Example 5A gastric cancer auxiliary diagnostic kit is prepared, wherein the kit contains a batch of SNPs specific amplification primers and specific probe primers (see Table 2), and further comprises reagents commonly used in PCR, such as Taq enzyme, dNTP mixed solution, deionized water and the like. Standard and control. Compared with other diagnostic instruments, the kit has the advantages of simplicity, high stability and capability of predicting disease states and genetic risks. Therefore, the kit is recommended to be put into clinical work, and decision basis is provided for screening and intervention of high-risk groups of gastric cancer.
TABLE 1 basic information of 42 gastric cancer Risk-associated SNPs loci
a Based on NCBI genome build (hg 19); b a reference/effector allele; c from thousand genome project data (china + japan); d LASSO regression coefficient
TABLE 2 typing primer information of 42 stomach cancer Risk-associated SNPs loci
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Note that: f: forward Primer, upstream Primer; r: reverse Primer, downstream Primer; FAM and HEX: fluorescent signals of different alleles.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Sequence listing
<110> university of Nanjing medical science
<120> genetic molecular marker related to gastric cancer auxiliary diagnosis and application thereof
<160> 168
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggaagaggct gcactgtcag a 21
<210> 3
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tctgggcctc tatgga 16
<210> 4
<211> 15
<212> DNA
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<400> 4
tctgggcctc tacgg 15
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cggatggtga ttccttgatt tt 22
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
agttcattca ctttgtgctt ctacaac 27
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
aattcctagc taccagaac 19
<210> 8
<211> 19
<212> DNA
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<400> 8
aattcctagc taacagaac 19
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gtgtgccagg catgcttcta 20
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gactgctcgg gttcaaatct tag 23
<210> 11
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
agaaaggcta aatgattt 18
<210> 12
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
agaaaggcta aatgactt 18
<210> 13
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctacaattat tttcacaggc tcaattct 28
<210> 14
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gaacattcca acatttgatc aacag 25
<210> 15
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
aagtggcatt tctat 15
<210> 16
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
agaagtggaa tttcta 16
<210> 17
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gcgtataaaa cacagtgcca attt 24
<210> 18
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
agcaaaccac tgacaccatc at 22
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
aataccaaat atacaaattt 20
<210> 20
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
aaattaaaat accaaatatg ca 22
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
tggccctgtt cctgtgaac 19
<210> 22
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gatccatctg caaacaaaat gagt 24
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
agaatctcat tttccctaaa 20
<210> 24
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
atctcatttc ccctaaaa 18
<210> 25
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cctacacggt ctatgttgcc acta 24
<210> 26
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gatccatggg ccaaatcct 19
<210> 27
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
aagcagccat aggtga 16
<210> 28
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
cagccatagg cgat 14
<210> 29
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
tgaagaagaa ccaatgtgtt gaaaa 25
<210> 30
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
aaatttgcaa tcagagaaaa gcgtat 26
<210> 31
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
tctgacaacc atctta 16
<210> 32
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
tctgacaaca atctt 15
<210> 33
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tgtccatgtg ttcccatttt tc 22
<210> 34
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
agacgtcaca ggttctccct tataa 25
<210> 35
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
tccccccagt aatc 14
<210> 36
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
tccccccaat aatc 14
<210> 37
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
acctgtgcaa atagatgcca att 23
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
ccattcaggc tcaggtttca 20
<210> 39
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
tcaaaaaatc aattcgaac 19
<210> 40
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
aaatcaattc aaacattgc 19
<210> 41
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gctgaggagg cagaggttac a 21
<210> 42
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
ccaaaattag tgcctttaag aaattcc 27
<210> 43
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ctaggtgaca gcgtga 16
<210> 44
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
cctaggtgac agagtg 16
<210> 45
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
ctctaccttc atagggcttt ttgg 24
<210> 46
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
tggtgggtaa aagagtccac tga 23
<210> 47
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
cccatcacaa aac 13
<210> 48
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
cccatcaaaa aaccaga 17
<210> 49
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
caccaaccaa caggatctaa ttga 24
<210> 50
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
gatccagcat ctggtctatg ttgt 24
<210> 51
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
cacacattca agtgc 15
<210> 52
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
agaatacaca cacactca 18
<210> 53
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
aattcctact gccatgacac taacac 26
<210> 54
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
gaatgaaagc acatgatgaa gaaga 25
<210> 55
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
acatccgtag ctcca 15
<210> 56
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
acatccgtag cgcca 15
<210> 57
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
ccaaatagct cagatgaaaa agtaacc 27
<210> 58
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
tttgtactct gtcctttcat tcctagag 28
<210> 59
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
tgtatacaaa tttcaggatc 20
<210> 60
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
tatacaaact tcaggatcc 19
<210> 61
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
tcttgctgtt tgtatggaat gttttt 26
<210> 62
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
gtggatgtcc tgctttttaa gga 23
<210> 63
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
cacaaatagg atctcggt 18
<210> 64
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
attcacaaat aggatctagg t 21
<210> 65
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
cttgccttga gcagaaaatg tg 22
<210> 66
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
ttccctgtct gccttgtgag a 21
<210> 67
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
ctctagcccc agagag 16
<210> 68
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
tctagcccca gaaag 15
<210> 69
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
gcagcctgaa gcagtcttct atc 23
<210> 70
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
ctctgacctt gacaagagga gtttc 25
<210> 71
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
ccactcccgt tagg 14
<210> 72
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
cactcccatt aggc 14
<210> 73
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
gcgtataaaa cacagtgcca attt 24
<210> 74
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
agcaaaccac tgacaccatc at 22
<210> 75
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
aataccaaat atacaaattt 20
<210> 76
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
aaattaaaat accaaatatg ca 22
<210> 77
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
tctagctaaa aacagagctg aagtcatt 28
<210> 78
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
ttatggtctg cagaaggatt tttg 24
<210> 79
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
aaaaaatccc ctttcgtc 18
<210> 80
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
aaaaaatccc ctttcatc 18
<210> 81
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
cagctcctga accagctcta gtc 23
<210> 82
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
agagggatct ggaatctcaa cct 23
<210> 83
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
ccatgcggtt tgg 13
<210> 84
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
ccatgcggtc tgg 13
<210> 85
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
atcactatcc ttgctacaga atccc 25
<210> 86
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 86
gccctgtgac cttgaatgag tc 22
<210> 87
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
cacttttcac tgaggaag 18
<210> 88
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 88
cacttttcat tgaggaag 18
<210> 89
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
ccagccttgc cttttcca 18
<210> 90
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
tgagcttctg tacaccacca cat 23
<210> 91
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
ctttgcatat ggttcag 17
<210> 92
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
tttgcatacg gttcaga 17
<210> 93
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
catagactgc tcccataagt atttcct 27
<210> 94
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
tttgaccacg gatgaagaat tct 23
<210> 95
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 95
ccacaattga ccttc 15
<210> 96
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 96
cacaattgac attctccaga 20
<210> 97
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 97
aacaaaaatc gcaagagtac caact 25
<210> 98
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 98
tgtgtctaaa ctcatcaaat ggtatgc 27
<210> 99
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 99
cttccacttt tttagggc 18
<210> 100
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 100
cttccacttt tttagagct 19
<210> 101
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 101
gggaagcaca cagtcctcat att 23
<210> 102
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 102
cccagcatgc agcactgat 19
<210> 103
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 103
agggccccag gtgg 14
<210> 104
<211> 13
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 104
ccccaggcgg ctg 13
<210> 105
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 105
caggagacag ccttgtagaa ctga 24
<210> 106
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 106
caagcctata agacaacctt cttgtg 26
<210> 107
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 107
tttgccctat cacgaaa 17
<210> 108
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 108
ttgccctatc acaaaa 16
<210> 109
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 109
catggaaaca gaagcccaaa g 21
<210> 110
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 110
ttcccatatc catcatctga ataaaa 26
<210> 111
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 111
aagcataaga tctgggtgt 19
<210> 112
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 112
aagcataaaa tctgggtgta g 21
<210> 113
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 113
atgttattta attttctcat gaggtaaatc a 31
<210> 114
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 114
actgacagca cttagacata ttcca 25
<210> 115
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 115
ttgatataat tttctttccc tcg 23
<210> 116
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 116
ttgatataat tttttttccc tcgc 24
<210> 117
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 117
cctctcctgg aatacagtta cctgtt 26
<210> 118
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 118
gcagatttca gctcagtatg agaaag 26
<210> 119
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 119
aggctctttc aatcgactgt 20
<210> 120
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 120
tctttcaatc tactgtct 18
<210> 121
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 121
tggagtaatg aagttttaag tgctca 26
<210> 122
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 122
gcatgactac actatattaa taaatgaaac cc 32
<210> 123
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 123
aactctttta cagcctaatg 20
<210> 124
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 124
aactctttta tagcctaatg 20
<210> 125
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 125
ggatgaacgg aatatacaaa gtattgaa 28
<210> 126
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 126
tgttccaccc acattaaaaa aatct 25
<210> 127
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 127
acaaagcaca gagcc 15
<210> 128
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 128
aaagcacaga accct 15
<210> 129
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 129
ggagaaaata agggaaagag ctctaga 27
<210> 130
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 130
cttgtcatgg aattagtaaa ggtgtgtat 29
<210> 131
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 131
caagtgcggt gcca 14
<210> 132
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 132
caagtgcgat gcca 14
<210> 133
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 133
aatcagggta aacatagtaa atgacatagg 30
<210> 134
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 134
cagcaggatg ttgtgatgta aactatt 27
<210> 135
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 135
tagactttct gttataccct c 21
<210> 136
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 136
caagctatag actttccgt 19
<210> 137
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 137
tttgtcattt gagctgttac tgtcttt 27
<210> 138
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 138
gatgagataa tgtgggtcaa tagatgag 28
<210> 139
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 139
ccataaggtg ctgagc 16
<210> 140
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 140
ttaccataaa gtgctgagc 19
<210> 141
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 141
ccccatgata tccctatttt ctcca 25
<210> 142
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 142
gagtggagat agaaggggag aattc 25
<210> 143
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 143
tatggagaat cggtactta 19
<210> 144
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 144
tatggagaat tggtacttac 20
<210> 145
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 145
tttgccaaaa tcagtcctgt ttt 23
<210> 146
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 146
cagaacttgt cggtggagtg aa 22
<210> 147
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 147
tgctggaact agctc 15
<210> 148
<211> 15
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 148
tgctggaact atctc 15
<210> 149
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 149
tgggctgaaa tgttgctttg 20
<210> 150
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 150
cagaagttcc ccgtcatatg c 21
<210> 151
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 151
agatggacaa tacgac 16
<210> 152
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 152
agatggacaa tacaac 16
<210> 153
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 153
cattttgcat cttcgtggtc atga 24
<210> 154
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 154
gatgatcatg ggagaggaag gtaaa 25
<210> 155
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 155
actctcccac tccact 16
<210> 156
<211> 14
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 156
actctcccgc tcca 14
<210> 157
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 157
cccaggctgg ctgcaa 16
<210> 158
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 158
tgctgtgaca gggtgaatga g 21
<210> 159
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 159
ctcagcaaat ccgtca 16
<210> 160
<211> 16
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 160
ctcagcaaat ccatca 16
<210> 161
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 161
cccaaagtgc tgggattaca g 21
<210> 162
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 162
ccttccagct ttatattagt gacaaca 27
<210> 163
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 163
ctacttctac ttattatctt t 21
<210> 164
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 164
tgctacttct acttattata tt 22
<210> 165
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 165
aataaaggca acatctcaac aaggt 25
<210> 166
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 166
gtcccttgct tgcctggat 19
<210> 167
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 167
acaaattgag gatacagtg 19
<210> 168
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 168
acaaattgag gacacagt 18
Claims (7)
1. A specific amplification primer of genetic molecular markers related to gastric cancer auxiliary diagnosis is characterized in that, genetic molecular markers related to gastric cancer auxiliary diagnosis are rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727 rs9341727, rs9341727 rs9341727, a combination of rs9341727 and rs 9341727;
The primer sequences of the specific amplification primer rs4460629 are SEQ ID NO.1 and SEQ ID NO.2;
primer sequences of rs4451581 are SEQ ID NO.5 and SEQ ID NO.6;
primer sequences of rs11802956 are SEQ ID NO.9 and SEQ ID NO.10;
primer sequences of rs236322 are SEQ ID NO.13 and SEQ ID NO.14;
primer sequences of rs11890919 are SEQ ID NO.17 and SEQ ID NO.18;
primer sequences of rs3771790 are SEQ ID NO.21 and SEQ ID NO.22;
primer sequences of rs7425197 are SEQ ID NO.25 and SEQ ID NO.26;
primer sequences of rs1403651 are SEQ ID NO.29 and SEQ ID NO.30;
primer sequences of rs4685983 are SEQ ID NO.33 and SEQ ID NO.34;
primer sequences of rs1504359 are SEQ ID NO.37 and SEQ ID NO.38;
primer sequences of rs2035057 are SEQ ID NO.41 and SEQ ID NO.42;
primer sequences of rs1870563 are SEQ ID NO.45 and SEQ ID NO.46;
primer sequences of rs1697952 are SEQ ID NO.49 and SEQ ID NO.50;
primer sequences of rs1564601 are SEQ ID NO.53 and SEQ ID NO.54;
the primer sequences of rs13195186 are SEQ ID NO.57 and SEQ ID NO.58;
primer sequences of rs9341727 are SEQ ID NO.61 and SEQ ID NO.62; primer sequences of rs3857490 are SEQ ID NO.65 and SEQ ID NO.66; primer sequences of rs6959127 are SEQ ID NO.69 and SEQ ID NO.70; primer sequences of rs4305854 are SEQ ID NO.73 and SEQ ID NO.74; primer sequences of rs1052969 are SEQ ID NO.77 and SEQ ID NO.78; primer sequences of rs9791835 are SEQ ID NO.81 and SEQ ID NO.82; primer sequences of rs10952328 are SEQ ID NO.85 and SEQ ID NO.86; primer sequences of rs7782998 are SEQ ID NO.89 and SEQ ID NO.90; primer sequences of rs656248 are SEQ ID NO.93 and SEQ ID NO.94;
Primer sequences of rs378363 are SEQ ID NO.97 and SEQ ID NO.98;
primer sequences of rs2460554 are SEQ ID NO.101 and SEQ ID NO.102; primer sequences of rs4751153 are SEQ ID NO.105 and SEQ ID NO.106; primer sequences of rs9329332 are SEQ ID NO.109 and SEQ ID NO.110; primer sequences of rs10777647 are SEQ ID NO.113 and SEQ ID NO.114; primer sequences of rs35326083 are SEQ ID NO.117 and SEQ ID NO.118; primer sequences of rs17027488 are SEQ ID NO.121 and SEQ ID NO.122; primer sequences of rs319284 are SEQ ID NO.125 and SEQ ID NO.126; primer sequences of rs2225046 are SEQ ID NO.129 and SEQ ID NO.130; primer sequences of rs10483947 are SEQ ID NO.133 and SEQ ID NO.134; primer sequences of rs7168752 are SEQ ID NO.137 and SEQ ID NO.138; primer sequences of rs875339 are SEQ ID NO.141 and SEQ ID NO.142; primer sequences of rs3093315 are SEQ ID NO.145 and SEQ ID NO.146; primer sequences of rs918655 are SEQ ID NO.149 and SEQ ID NO.150; primer sequences of rs348793 are SEQ ID NO.153 and SEQ ID NO.154; primer sequences of rs17000838 are SEQ ID NO.157 and SEQ ID NO.158; primer sequences of rs1034420 are SEQ ID NO.161 and SEQ ID NO.162; primer sequences of rs2019061 are SEQ ID NO.165 and SEQ ID NO.166.
2. A specific probe primer of a genetic molecular marker related to gastric cancer auxiliary diagnosis is characterized in that the primer sequence of the specific probe primer rs4460629 is SEQ ID NO.3 and SEQ ID NO.4;
primer sequences of rs4451581 are SEQ ID NO.7 and SEQ ID NO.8;
primer sequences of rs11802956 are SEQ ID NO.11 and SEQ ID NO.12;
primer sequences of rs236322 are SEQ ID NO.15 and SEQ ID NO.16;
primer sequences of rs11890919 are SEQ ID NO.19 and SEQ ID NO.20;
primer sequences of rs3771790 are SEQ ID NO.23 and SEQ ID NO.24;
primer sequences of rs7425197 are SEQ ID NO.27 and SEQ ID NO.28;
primer sequences of rs1403651 are SEQ ID NO.31 and SEQ ID NO.32;
primer sequences of rs4685983 are SEQ ID NO.35 and SEQ ID NO.36;
primer sequences of rs1504359 are SEQ ID NO.39 and SEQ ID NO.40;
primer sequences of rs2035057 are SEQ ID NO.43 and SEQ ID NO.44;
primer sequences of rs1870563 are SEQ ID NO.47 and SEQ ID NO.48;
primer sequences of rs1697952 are SEQ ID NO.51 and SEQ ID NO.52;
primer sequences of rs1564601 are SEQ ID NO.55 and SEQ ID NO.56;
primer sequences of rs13195186 are SEQ ID NO.59 and SEQ ID NO.60;
primer sequences of rs9341727 are SEQ ID NO.63 and SEQ ID NO.64;
Primer sequences of rs3857490 are SEQ ID NO.67 and SEQ ID NO.68;
primer sequences of rs6959127 are SEQ ID NO.71 and SEQ ID NO.72;
primer sequences of rs4305854 are SEQ ID NO.75 and SEQ ID NO.76;
primer sequences of rs1052969 are SEQ ID NO.79 and SEQ ID NO.80;
primer sequences of rs9791835 are SEQ ID NO.83 and SEQ ID NO.84;
primer sequences of rs10952328 are SEQ ID NO.87 and SEQ ID NO.88;
primer sequences of rs7782998 are SEQ ID NO.91 and SEQ ID NO.92;
primer sequences of rs656248 are SEQ ID NO.95 and SEQ ID NO.96;
primer sequences of rs378363 are SEQ ID NO.99 and SEQ ID NO.100;
primer sequences of rs2460554 are SEQ ID NO.103 and SEQ ID NO.104;
primer sequences of rs4751153 are SEQ ID NO.107 and SEQ ID NO.108;
primer sequences of rs9329332 are SEQ ID NO.111 and SEQ ID NO.112;
primer sequences of rs10777647 are SEQ ID NO.115 and SEQ ID NO.116;
primer sequences of rs35326083 are SEQ ID NO.119 and SEQ ID NO.120;
primer sequences of rs17027488 are SEQ ID NO.123 and SEQ ID NO.124;
primer sequences of rs319284 are SEQ ID NO.127 and SEQ ID NO.128;
primer sequences of rs2225046 are SEQ ID NO.131 and SEQ ID NO.132;
primer sequences of rs10483947 are SEQ ID NO.135 and SEQ ID NO.136;
Primer sequences of rs7168752 are SEQ ID NO.139 and SEQ ID NO.140;
primer sequences of rs875339 are SEQ ID NO.143 and SEQ ID NO.144;
primer sequences of rs3093315 are SEQ ID NO.147 and SEQ ID NO.148;
primer sequences of rs918655 are SEQ ID NO.151 and SEQ ID NO.152;
primer sequences of rs348793 are SEQ ID NO.155 and SEQ ID NO.156;
primer sequences of rs17000838 are SEQ ID NO.159 and SEQ ID NO.160;
primer sequences of rs1034420 are SEQ ID NO.163 and SEQ ID NO.164;
primer sequences of rs2019061 are SEQ ID NO.167 and SEQ ID NO.168.
3. Use of a specific amplification primer of a genetic molecular marker related to gastric cancer auxiliary diagnosis according to claim 1 for preparing a gastric cancer auxiliary diagnosis kit.
4. Use of a specific probe primer of a genetic molecular marker related to gastric cancer auxiliary diagnosis according to claim 2 for preparing a gastric cancer auxiliary diagnosis kit.
5. The gastric cancer auxiliary diagnosis kit is used for detecting the gene types of rs4460629, rs4451581, rs11802956, rs236322, rs11890919, rs3771790, rs7425197, rs1403651, rs4685983, rs1504359, rs2035057, rs1870563, rs1697952, rs1564601, rs13195186, rs9341727, rs3857490, rs6959127, rs4305854, rs1052969, rs9791835, rs10952328, rs7782998, rs656248, rs378363, rs2460554, rs4751153, rs9329332, rs10777647, rs35326083, rs17027488, rs319284, rs2225046, rs10483947, rs7168752, rs875339, rs3093315, rs918655, rs348793, rs17000838, rs1034420 and rs2019061 in peripheral blood DNA;
The gastric cancer auxiliary diagnosis kit contains a specific amplification primer and a specific probe primer;
the primer sequences of the specific amplification primer rs4460629 are SEQ ID NO.1 and SEQ ID NO.2;
primer sequences of rs4451581 are SEQ ID NO.5 and SEQ ID NO.6;
primer sequences of rs11802956 are SEQ ID NO.9 and SEQ ID NO.10;
primer sequences of rs236322 are SEQ ID NO.13 and SEQ ID NO.14;
primer sequences of rs11890919 are SEQ ID NO.17 and SEQ ID NO.18;
primer sequences of rs3771790 are SEQ ID NO.21 and SEQ ID NO.22;
primer sequences of rs7425197 are SEQ ID NO.25 and SEQ ID NO.26;
primer sequences of rs1403651 are SEQ ID NO.29 and SEQ ID NO.30;
primer sequences of rs4685983 are SEQ ID NO.33 and SEQ ID NO.34;
primer sequences of rs1504359 are SEQ ID NO.37 and SEQ ID NO.38;
primer sequences of rs2035057 are SEQ ID NO.41 and SEQ ID NO.42;
primer sequences of rs1870563 are SEQ ID NO.45 and SEQ ID NO.46;
primer sequences of rs1697952 are SEQ ID NO.49 and SEQ ID NO.50;
primer sequences of rs1564601 are SEQ ID NO.53 and SEQ ID NO.54;
the primer sequences of rs13195186 are SEQ ID NO.57 and SEQ ID NO.58;
primer sequences of rs9341727 are SEQ ID NO.61 and SEQ ID NO.62;
Primer sequences of rs3857490 are SEQ ID NO.65 and SEQ ID NO.66;
primer sequences of rs6959127 are SEQ ID NO.69 and SEQ ID NO.70;
primer sequences of rs4305854 are SEQ ID NO.73 and SEQ ID NO.74; primer sequences of rs1052969 are SEQ ID NO.77 and SEQ ID NO.78; primer sequences of rs9791835 are SEQ ID NO.81 and SEQ ID NO.82; primer sequences of rs10952328 are SEQ ID NO.85 and SEQ ID NO.86; primer sequences of rs7782998 are SEQ ID NO.89 and SEQ ID NO.90; primer sequences of rs656248 are SEQ ID NO.93 and SEQ ID NO.94;
primer sequences of rs378363 are SEQ ID NO.97 and SEQ ID NO.98;
primer sequences of rs2460554 are SEQ ID NO.101 and SEQ ID NO.102; primer sequences of rs4751153 are SEQ ID NO.105 and SEQ ID NO.106; primer sequences of rs9329332 are SEQ ID NO.109 and SEQ ID NO.110; primer sequences of rs10777647 are SEQ ID NO.113 and SEQ ID NO.114; primer sequences of rs35326083 are SEQ ID NO.117 and SEQ ID NO.118; primer sequences of rs17027488 are SEQ ID NO.121 and SEQ ID NO.122; primer sequences of rs319284 are SEQ ID NO.125 and SEQ ID NO.126; primer sequences of rs2225046 are SEQ ID NO.129 and SEQ ID NO.130; primer sequences of rs10483947 are SEQ ID NO.133 and SEQ ID NO.134; primer sequences of rs7168752 are SEQ ID NO.137 and SEQ ID NO.138; primer sequences of rs875339 are SEQ ID NO.141 and SEQ ID NO.142; primer sequences of rs3093315 are SEQ ID NO.145 and SEQ ID NO.146; primer sequences of rs918655 are SEQ ID NO.149 and SEQ ID NO.150; primer sequences of rs348793 are SEQ ID NO.153 and SEQ ID NO.154; primer sequences of rs17000838 are SEQ ID NO.157 and SEQ ID NO.158; primer sequences of rs1034420 are SEQ ID NO.161 and SEQ ID NO.162; the primer sequences of rs2019061 are SEQ ID NO.165 and SEQ ID NO.166; the primer sequence of the specific probe primer rs4460629 is SEQ ID NO.3 and SEQ ID NO.4;
Primer sequences of rs4451581 are SEQ ID NO.7 and SEQ ID NO.8; primer sequences of rs11802956 are SEQ ID NO.11 and SEQ ID NO.12; primer sequences of rs236322 are SEQ ID NO.15 and SEQ ID NO.16; primer sequences of rs11890919 are SEQ ID NO.19 and SEQ ID NO.20; primer sequences of rs3771790 are SEQ ID NO.23 and SEQ ID NO.24; primer sequences of rs7425197 are SEQ ID NO.27 and SEQ ID NO.28; primer sequences of rs1403651 are SEQ ID NO.31 and SEQ ID NO.32; primer sequences of rs4685983 are SEQ ID NO.35 and SEQ ID NO.36; primer sequences of rs1504359 are SEQ ID NO.39 and SEQ ID NO.40; primer sequences of rs2035057 are SEQ ID NO.43 and SEQ ID NO.44; primer sequences of rs1870563 are SEQ ID NO.47 and SEQ ID NO.48; primer sequences of rs1697952 are SEQ ID NO.51 and SEQ ID NO.52; primer sequences of rs1564601 are SEQ ID NO.55 and SEQ ID NO.56; primer sequences of rs13195186 are SEQ ID NO.59 and SEQ ID NO.60; primer sequences of rs9341727 are SEQ ID NO.63 and SEQ ID NO.64; primer sequences of rs3857490 are SEQ ID NO.67 and SEQ ID NO.68; primer sequences of rs6959127 are SEQ ID NO.71 and SEQ ID NO.72; primer sequences of rs4305854 are SEQ ID NO.75 and SEQ ID NO.76; primer sequences of rs1052969 are SEQ ID NO.79 and SEQ ID NO.80; primer sequences of rs9791835 are SEQ ID NO.83 and SEQ ID NO.84; primer sequences of rs10952328 are SEQ ID NO.87 and SEQ ID NO.88; primer sequences of rs7782998 are SEQ ID NO.91 and SEQ ID NO.92; primer sequences of rs656248 are SEQ ID NO.95 and SEQ ID NO.96; primer sequences of rs378363 are SEQ ID NO.99 and SEQ ID NO.100; primer sequences of rs2460554 are SEQ ID NO.103 and SEQ ID NO.104; primer sequences of rs4751153 are SEQ ID NO.107 and SEQ ID NO.108; primer sequences of rs9329332 are SEQ ID NO.111 and SEQ ID NO.112;
Primer sequences of rs10777647 are SEQ ID NO.115 and SEQ ID NO.116;
primer sequences of rs35326083 are SEQ ID NO.119 and SEQ ID NO.120;
primer sequences of rs17027488 are SEQ ID NO.123 and SEQ ID NO.124;
primer sequences of rs319284 are SEQ ID NO.127 and SEQ ID NO.128;
primer sequences of rs2225046 are SEQ ID NO.131 and SEQ ID NO.132;
primer sequences of rs10483947 are SEQ ID NO.135 and SEQ ID NO.136;
primer sequences of rs7168752 are SEQ ID NO.139 and SEQ ID NO.140;
primer sequences of rs875339 are SEQ ID NO.143 and SEQ ID NO.144;
primer sequences of rs3093315 are SEQ ID NO.147 and SEQ ID NO.148;
primer sequences of rs918655 are SEQ ID NO.151 and SEQ ID NO.152;
primer sequences of rs348793 are SEQ ID NO.155 and SEQ ID NO.156;
primer sequences of rs17000838 are SEQ ID NO.159 and SEQ ID NO.160;
primer sequences of rs1034420 are SEQ ID NO.163 and SEQ ID NO.164;
primer sequences of rs2019061 are SEQ ID NO.167 and SEQ ID NO.168.
6. The gastric cancer auxiliary diagnostic kit of claim 4 or 5, wherein the kit further comprises PCR reagents.
7. The gastric cancer auxiliary diagnostic kit according to claim 6, wherein the PCR reagent is Taq enzyme, dNTP mixed solution, deionized water, standard substance and reference substance.
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| CN104278085A (en) * | 2014-09-15 | 2015-01-14 | 南京医科大学 | Early stomach cancer assisted diagnosis related SNP (single-nucleotide polymorphism) marker and application thereof |
| CN107254531A (en) * | 2017-06-28 | 2017-10-17 | 南京医科大学 | The genetic biomarkers thing of early onset colorectal cancer auxiliary diagnosis and its application |
| WO2019168478A1 (en) * | 2018-03-01 | 2019-09-06 | Agency For Science, Technology And Research | A method of determining a risk of cancer |
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| CN104278085A (en) * | 2014-09-15 | 2015-01-14 | 南京医科大学 | Early stomach cancer assisted diagnosis related SNP (single-nucleotide polymorphism) marker and application thereof |
| CN107254531A (en) * | 2017-06-28 | 2017-10-17 | 南京医科大学 | The genetic biomarkers thing of early onset colorectal cancer auxiliary diagnosis and its application |
| WO2019168478A1 (en) * | 2018-03-01 | 2019-09-06 | Agency For Science, Technology And Research | A method of determining a risk of cancer |
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