Application based on helicobacter pylori mononucleotide site in excrement

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to application based on helicobacter pylori mononucleotide sites in excrement. According to the invention, through a GWAS technology, helicobacter pylori specific SNP sites (s1625711 and s407233) significantly related to gastric cancer occurrence are screened from gastric cancer patients and control people; then, in a portable and noninvasive excrement sample of a patient, the mutation condition of the SNP site is detected by utilizing a molecular biological detection means, so that early noninvasive screening and risk assessment of gastric cancer are performed on the helicobacter pylori infected patient. According to the invention, a molecular genetics marker is creatively combined with portable noninvasive detection, so that not only can the sensitivity and accessibility of early screening of gastric cancer be effectively improved, but also an important basis can be provided for later precise treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and in particular to an application based on a single nucleotide site of Helicobacter pylori in feces. Background Art

[0002] Gastric cancer is a common malignant tumor with high morbidity and mortality, and early detection and treatment are crucial to improving the prognosis of patients. At present, there are various methods for early diagnosis and screening of gastric cancer. Gastroscopy and biopsy for pathological examination are currently the gold standard for diagnosing gastric cancer, but the operation cost is high and the patient tolerance and compliance are poor. The sensitivity of serological tests such as pepsinogen and gastrin is low and is easily affected by diet, environment, etc. In clinical practice, the diversity changes of fecal flora are used to indirectly assess the risk of gastrointestinal tumors. The principle of this method is that when a tumor occurs, the composition and diversity of the intestinal flora will change. However, the diversity of the intestinal flora is affected by many factors, such as diet, medication, lifestyle, etc., and there are large individual differences and dynamic changes. Therefore, the specificity and sensitivity of tumor screening based solely on changes in the flora are limited, and the differential diagnosis ability is insufficient.

[0003] Helicobacter pylori infection is recognized as one of the main risk factors for gastric cancer. Helicobacter pylori is closely associated with a variety of digestive tract diseases such as gastritis and peptic ulcers, and is closely related to about 80% of gastric cancer cases. As early as 1994, it was listed as a Class I carcinogen by the World Health Organization. Existing technologies mostly focus on detecting the presence or absence of Helicobacter pylori, but fail to further utilize the molecular characteristics of Helicobacter pylori itself for precise screening. At the same time, existing conventional screening methods for gastric cancer, such as gastroscopy, have certain disadvantages such as trauma, complexity and high cost.

[0004] Therefore, there is an urgent need to develop a new non-invasive method for early screening of gastric cancer based on Helicobacter pylori-specific molecular markers to improve the sensitivity and specificity of screening and reduce the pain and economic burden of patients, thereby achieving precision medicine and personalized diagnosis and treatment. Summary of the invention

[0005] In order to solve the problems of certain trauma, complexity and high cost in the conventional screening of Helicobacter pylori for gastric cancer, the purpose of the present invention is to provide an application based on the single nucleotide sites of Helicobacter pylori in feces. The present invention uses the whole genome association analysis (GWAS) technology to screen out Helicobacter pylori-specific SNP sites that are significantly associated with gastric cancer from gastric cancer patients and control populations; then, in the patient's portable and non-invasive fecal samples, the mutation of the SNP site is detected by molecular biological detection methods, so as to perform early non-invasive screening and risk assessment of gastric cancer in patients infected with Helicobacter pylori.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The first object of the present invention is to provide a kit for gastric cancer screening, the kit comprising: a primer set for amplifying Helicobacter pylori single nucleotide sites s1625711 and s407233, and a probe for specifically detecting the wild type and mutant genotypes of Helicobacter pylori single nucleotide sites s1625711 and s407233;

[0008] Among them, the s1625711 site is located in the Helicobacter pylori tRNA ligase gene, and the mutation of the s1625711 site causes the methionine at position 744 to become valine; the s407233 site is located on the gene HP0396, and the mutation of the s407233 site causes the lysine at position 10 to become glutamate.

[0009] In one embodiment of the present invention, the kit is a kit using ex vivo feces as a sample, and the kit further contains a reagent for extracting total DNA from the feces sample.

[0010] In one embodiment of the present invention, the primer set for amplifying the single nucleotide site s1625711 of Helicobacter pylori includes F 1625711 and R 1625711 ,

[0011] F 1625711 :5'-CAAGCCTATAGCGATCGATGAA-3';

[0012] R 1625711 :5'-TCAATTCTGCACGCCTTTTG-3';

[0013] The primer set for amplifying the single nucleotide site s407233 of Helicobacter pylori includes F 407233 and R 407233 , F 407233 :5'-GGTTATTTAAGGTAGTTGGATGCG-3';

[0014] R 407233 :5'-AATAAAAGGGCTTTTGCCCCCATTA-3';

[0015] The probe used to specifically detect the wild type of Helicobacter pylori locus s1625711 is as follows:

[0016] F 1625711w : 5'-CGCTTTGATGGAAG-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0017] The probe used to specifically detect the mutant genotype of Helicobacter pylori locus s1625711 is as follows:

[0018] F 1625711m： 5'-ACGCTTTAGTGGAAG-3', with HEX modification at the 5' end and mgb and NFQ modifications at the 3' end;

[0019] Probe for specific detection of wild-type Helicobacter pylori locus s407233:

[0020] F 407233w : 5'-CTTTTAAAAAAGCATGA-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0021] Probe for specific detection of Helicobacter pylori locus s407233 mutation genotype:

[0022] F 407233m： 5'-TTAAAAGAGCATGATGA-3', wherein the 5' end has HEX modification, and the 3' end has mgb and NFQ modifications in sequence.

[0023] In one embodiment of the present invention, it includes a PCR amplification reaction system and a PCR product purification system.

[0024] The second object of the present invention is to provide a kit for evaluating the prognosis or judging the recurrence and metastasis after treatment of gastric cancer, the kit comprising: a primer set for amplifying the single nucleotide sites s1625711 and s407233 of Helicobacter pylori, and a probe for specifically detecting the wild type and mutant genotypes of the single nucleotide sites s1625711 and s407233 of Helicobacter pylori;

[0025] Among them, the s1625711 site is located in the Helicobacter pylori tRNA ligase gene, and the mutation of the s1625711 site causes the methionine at position 744 to become valine; the s407233 site is located on the gene HP0396, and the mutation of the s407233 site causes the lysine at position 10 to become glutamate.

[0026] In one embodiment of the present invention, the kit is a kit using ex vivo feces as a sample, and the kit further contains a reagent for extracting total DNA from the feces sample.

[0027] In one embodiment of the present invention, the primer set for amplifying the single nucleotide site s1625711 of Helicobacter pylori includes F 1625711 and R 1625711 ,

[0028] F 1625711 :5'-CAAGCCTATAGCGATCGATGAA-3';

[0029] R 1625711 :5'-TCAATTCTGCACGCCTTTTG-3';

[0030] The primer set for amplifying the single nucleotide site s407233 of Helicobacter pylori includes F 407233 and R 407233 , F 407233 :5'-GGTTATTTAAGGTAGTTGGATGCG-3';

[0031] R 407233 :5'-AATAAAAGGGCTTTTGCCCCCATTA-3';

[0032] The probe used to specifically detect the wild type of Helicobacter pylori locus s1625711 is as follows:

[0033] F 1625711w : 5'-CGCTTTGATGGAAG-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0034] The probe used to specifically detect the mutant genotype of Helicobacter pylori locus s1625711 is as follows:

[0035] F 1625711m： 5'-ACGCTTTAGTGGAAG-3', with HEX modification at the 5' end and mgb and NFQ modifications at the 3' end;

[0036] Probe for specific detection of wild-type Helicobacter pylori locus s407233:

[0037] F 407233w : 5'-CTTTTAAAAAAGCATGA-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0038] Probe for specific detection of Helicobacter pylori locus s407233 mutation genotype:

[0039] F 407233m： 5'-TTAAAAGAGCATGATGA-3', wherein the 5' end has HEX modification, and the 3' end has mgb and NFQ modifications in sequence.

[0040] In one embodiment of the present invention, it includes a PCR amplification reaction system and a PCR product purification system.

[0041] The third object of the present invention is to provide a substance for inhibiting the mutation of a single nucleotide site of Helicobacter pylori for use in the preparation of a vaccine for preventing early gastric cancer, wherein the single nucleotide site is selected from one or both of s1625711 and s407233.

[0042] The fourth object of the present invention is to provide a vaccine for preventing early gastric cancer, wherein the vaccine contains a substance that inhibits the mutation of a single nucleotide site of Helicobacter pylori;

[0043] The single nucleotide site is selected from one or both of s1625711 and s407233.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) Higher screening sensitivity and specificity: Compared with the existing technology that mainly relies on evaluating the dynamic changes of the overall fecal flora (which is affected by multiple factors and has relatively low specificity and sensitivity), the present invention uses GWAS to select Helicobacter pylori-specific SNP sites that are highly correlated with gastric cancer from a large amount of case and control data as markers, which can directly reflect the changes in the carcinogenic molecules of Helicobacter pylori in patients, thereby greatly improving the sensitivity and specificity of gastric cancer screening.

[0046] (2) Non-invasive and simple detection method: The present invention only needs to collect stool samples from patients, and then extract stool DNA and perform SNP site detection, avoiding the pain caused to patients by invasive examinations such as traditional gastroscopy; stool sampling is simple and non-invasive. Based on the scheme of the present invention, it can provide patients with a more comfortable and acceptable screening experience, and it is also conducive to promotion to large-scale population censuses and preliminary screening.

[0047] (3) Early detection of high-risk groups for gastric cancer: The present invention can accurately identify high-risk patients who carry mutations in specific SNP sites (s1625711 and / or s407233 sites) in Helicobacter pylori. Once patients carrying mutations in carcinogenic SNP sites are detected, further examination and treatment can be given in a timely manner, thereby facilitating early detection of gastric cancer lesions and improving treatment effects.

[0048] (4) Guiding individualized clinical treatment: The Helicobacter pylori SNP markers provided by the present invention can guide subsequent individualized treatment plans. Once a patient carrying a mutation in a carcinogenic SNP site (s1625711 and / or s407233 site) is detected, more accurate and effective drugs can be selected according to their pathogenic mechanism, such as antibiotics and enzyme inhibitors that specifically target Helicobacter pylori, to provide patients with more accurate treatment. At the same time, this information is also helpful in evaluating the patient's sensitivity to certain therapeutic drugs, providing a basis for clinical drug use.

[0049] In summary, the present invention innovatively combines molecular genetic markers with portable non-invasive detection, which can not only effectively improve the sensitivity and accessibility of early screening of gastric cancer, but also provide an important basis for later precision treatment. It has significant clinical application value and broad development prospects, which cannot be achieved by existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Manhattan plot of the distribution of SNP sites analyzed by GWAS;

[0051] Figure 2 It is the ROC curve diagram of the typing model in Example 1;

[0052] Figure 3 The ROC curve diagram of the typing model validated by the sequencing data of clinical isolates in Example 1. DETAILED DESCRIPTION

[0053] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0054] In the following examples, unless otherwise specified, the reagents are all commercially available reagents, and the detection means and methods used are all conventional detection means and methods in the art.

[0055] Example 1

[0056] A total of 226 gastric cancer isolates (i.e., Helicobacter pylori strains isolated from gastric cancer patients) and 1476 non-gastric cancer isolates (i.e., Helicobacter pylori strains isolated from non-gastric cancer patients) from the NCBI database were selected, of which a total of 5431 single nucleotide polymorphism (SNP) sites passed the pre-set quality control and filtering criteria (the obtained SNP data were filtered using PLINKv2.0 to exclude SNPs with an allele frequency of less than 5% or a data missing rate of more than 10%, the results are as follows Figure 1 shown).

[0057] Using gastric cancer and non-gastric cancer sources as phenotypes and different geographical locations as covariates, three models, fixed effect and random effect models (FarmCPU model), generalized linear model (GLC model) and linear mixed model (LMM model) were used for association analysis. Random forest tree and LASSO regression algorithm were used.

[0058] Specifically, three analysis models were implemented: FarmCPU model, GLM model, and LMM model. The top 10 most strongly correlated GWAS genes appeared in 10 genes with a frequency difference greater than 20% and a P value ≤ 10 -6Among the genes, the screening criteria are set as follows: the mutation rate of the site in both the modeling data and the verification data exceeds 50%, and the mutation difference exceeds 30%.

[0059] First, the first-ranked SNP site was determined: s1625711 (153 / 226 (67.70%) in cancer isolates, 163 / 1469 (11.04%) in controls, OR = 17.28); Next, the screening range was further expanded, the top 40 non-synonymous mutations were compared, and the second SNP site was finally determined: s407233 (126 / 226 (55.75%) in cancer isolates, 297 / 1469 (20.12%) in controls; OR = 5.05). A total of 2 key SNP sites were determined: s1625711 and s407233, as the final typing model. Both sites contain non-synonymous mutations.

[0060] Among them, the s1625711 site is located in the Helicobacter pylori tRNA ligase gene, and the mutation of the s1625711 site causes the methionine at position 744 to become valine; the s407233 site is located on the HP0396 gene, and the mutation of the s407233 site causes the lysine at position 10 to become glutamic acid (as shown in Table 1).

[0061] Table 1 Summary of information on s1625711 and s407233 sites

[0062]

[0063] Among them, site s1625711 mutation occurred in 153 (67.70%) gastric cancer isolates and 163 (11.04%) non-gastric cancer isolates; site s407233 mutation occurred in 126 (55.75%) gastric cancer isolates and 297 (20.12%) non-gastric cancer isolates.

[0064] According to whether the two SNP sites are mutated, the strains are divided into a high-risk group (mutation group) and a low-risk group (non-mutation group) for gastric cancer. The ROC curve is used to evaluate the model. The results are as follows: Figure 2 As shown, through Figure 2 It can be found that in the modeling group data, the AUC area of ​​the model is 0.793, the specificity is 0.890, and the sensitivity is 0.648.

[0065] Furthermore, in order to verify the effectiveness of the model, the whole genome sequencing data of clinical isolates was used to analyze and extract the mutation information of s1625711 and s407233 sites and verify it. Specifically, gastric mucosal samples from gastric cancer patients in the Department of Gastroenterology 1 of the Affiliated Tumor Hospital of Fudan University were collected, and a total of 19 gastric cancer-derived strains were isolated and cultured. Gastric mucosal samples under gastroscopy from outpatients in the Department of Gastroenterology of the Affiliated Oriental Hospital of Tongji University were collected, and a total of 22 non-gastric cancer-derived strains were isolated and cultured. In the gastric cancer isolates, the mutation positive rates of s1625711 and s407233 sites were 94.74% (18 strains) and 100.00% (19 strains), respectively, which increased by about 50% compared with the non-gastric cancer isolates (the mutation positive rates of s1625711 and s407233 sites were 31.82% (7 strains) and 36.36% (8 strains), respectively.

[0066] Using the same typing method, 74% of the gastric cancer group were classified as high-risk strains for gastric cancer (mutations at s1625711 and / or s407233 sites), while 32% of the non-gastric cancer group were high-risk strains for gastric cancer (mutations at s1625711 and / or s407233 sites). The difference between the two groups was statistically significant (P = 0.018). The ROC curve was also used to evaluate the model. The results are as follows: Figure 3 As shown, through Figure 3 It can be found that the AUC area of ​​the model is 0.945, the specificity is 0.909, and the sensitivity is 0.947.

[0067] The method for extracting total DNA from fecal samples for model validation is as follows:

[0068] Collect fresh stool samples from patients who need to be screened. After the fresh samples are collected, they are immediately placed in a -80℃ ultra-low temperature refrigerator for freezing and preservation to avoid DNA degradation. Use the stool DNA extraction kit TIANGEN's fecal Genomic DNA Extraction Kit (DP328) that meets national standards and standard operating procedures to extract total DNA as soon as possible.

[0069] The specific steps are: first weigh an appropriate amount of fecal sample, add DNA lysis solution and protease and other reagents, shake on a shaker to mix and lyse it thoroughly; then heat and incubate for a period of time; then use a high salt concentration inorganic compound solution to perform protein salting out on the cleavage mixture to remove large molecular protein impurities; then centrifuge, elute, purify and other steps to finally obtain high-quality fecal total DNA.

[0070] Primer Express V 3.0.1 was used to design point mutation primers, and Primer-BLAST (http: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) and MFEprimer V 4.0 (https: / / m4.igenetech.com / spec / ) were used to verify the specificity of the designed primers.

[0071] Among them, the primer set designed based on s1625711 is as follows:

[0072] F 1625711 (SEQ ID NO.1): 5'-CAAGCCTATAGCGATCGATGAA-3';

[0073] R 1625711 (SEQ ID NO.2): 5'-TCAATTCTGCACGCCTTTTG-3';

[0074] The probe used to specifically detect the wild type of Helicobacter pylori locus s1625711 is as follows:

[0075] F 1625711w (SEQ ID NO. 3): 5'-CGCTTTGATGGAAG-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0076] that is, 5′-FAM-CGCTTTGATGGAAG-mgb-NFQ-3′;

[0077] The probe used to specifically detect the mutant genotype of Helicobacter pylori locus s1625711 is as follows:

[0078] F 1625711m (SEQ ID NO. 4): 5'-ACGCTTTAGTGGAAG-3', wherein the 5' end has a HEX modification, and the 3' end has mgb and NFQ modifications in sequence;

[0079] that is, 5′-HEX-ACGCTTTAGTGGAAG-mgb-NFQ-3′;

[0080] The primer sets designed based on s407233 are as follows:

[0081] The primer set for amplifying the single nucleotide site s407233 of Helicobacter pylori includes F 407233 and R 407233 , F 407233(SEQ ID NO.5): 5'-GGTTATTTAAGGTAGTTGGATCGCG-3';

[0082] R 407233 (SEQ ID NO. 6): 5'-AATAAAAGGGCTTTTGCCCCCATTA-3'.

[0083] Probe for specific detection of wild-type Helicobacter pylori locus s407233:

[0084] F 407233w (SEQ ID NO. 7): 5'-CTTTTAAAAAAGCATGA-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence;

[0085] that is, 5′-FAM-CTTTTAAAAAAGCATGA-mgb-NFQ-3′;

[0086] Probe for specific detection of Helicobacter pylori locus s407233 mutation genotype:

[0087] F 407233m (SEQ ID NO. 8): 5'-TTAAAAGAGCATGATGA-3', wherein the 5' end has a HEX modification, and the 3' end has mgb and NFQ modifications in sequence;

[0088] That is, 5'-HEX-TTAAAAGAGCATGATGA-mgb-NFQ-3'.

[0089] Taqman quantitative real-time fluorescence PCR (qPCR) was performed using ChamQ Geno-SNP Probe Master Mix (Vazyme, Shanghai), and the reaction program included pre-denaturation at 95°C for 30 seconds, followed by cycling: denaturation at 95°C for 10 seconds, annealing / extension at 60°C for 30 minutes, and 40 cycles. The experiment was performed on a real-time fluorescence PCR instrument (QuantStudio 6Thermo, USA). DNA extracted from strain 09142 (containing two SNPs mutations) was used as a positive control to verify the presence of the target sequence, while strain 26695 was used as a negative control.

[0090] A total of 473 gastric cancer patient samples diagnosed in the Department of Gastroenterology 1 of the Affiliated Cancer Hospital of Fudan University, and 290 samples from non-gastric cancer patients and healthy physical examination subjects from Shanghai Zhoupu Hospital, Shanghai Oriental Hospital, Shanghai Changzheng Hospital, and Anqing First People's Hospital of Anhui Medical University were collected as controls (details are shown in Table 2). Among the samples of tumor patients, 296 (62.58%) patients were positive for Helicobacter pylori by 16S rRNA PCR results to determine whether there was evidence of Helicobacter pylori genome in stool samples, and 177 (61.03%) of the non-gastric cancer samples (non-gastric cancer and healthy physical examination patients) were positive for Helicobacter pylori. The sequencing results showed that the mutation positive rate of s1625711 in fecal samples of tumor patients was 55.07% (163 cases), while in fecal samples of non-gastric cancer, the mutation positive rate of s1625711 was 19.21% (34 cases); the mutation positive rate of s407233 site in fecal samples of tumor patients was 44.59% (132 cases), while in non-gastric cancer samples, the mutation positive rate of s407233 was 20.34% (36 cases); the difference between the two groups was statistically significant.

[0091] Table 2 Analysis of sample collection

[0092]

[0093] In summary, based on the two specific SNP sites s1625711 and s407233 determined in the GWAS analysis, PCR primer pairs targeting the gene sequences near these two sites are designed, and the PCR reaction system and program are adjusted accordingly according to the length of the target fragment to be amplified; based on this, a multi-site SNP diagnostic kit can be further prepared. Specifically, a gene amplification instrument is used to perform SNP detection on the extracted fecal DNA, and then the PCR products are sequenced or based on high-resolution melting curve analysis and other methods to accurately identify whether the two SNP sites (s1625711 and s407233 sites) have mutated; if mutations are detected in the s1625711 and / or s407233 sites, it is determined that the patient has a high risk of gastric cancer and needs to seek medical treatment in time and undergo further examinations such as gastroscopy to clarify the diagnosis and formulate subsequent treatment plans.

[0094] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A kit for gastric cancer screening, characterized in that: The kit contains: a primer set for amplifying the single nucleotide sites s1625711 and s407233 of Helicobacter pylori, and a probe for specifically detecting the wild type and mutant genotypes of the single nucleotide sites s1625711 and s407233 of Helicobacter pylori; Among them, the s1625711 site is located in the Helicobacter pylori tRNA ligase gene, and the mutation of the s1625711 site causes the methionine at position 744 to become valine; the s407233 site is located on the gene HP0396, and the mutation of the s407233 site causes the lysine at position 10 to become glutamate.

2. The kit for gastric cancer screening according to claim 1, characterized in that: The kit is a kit using in vitro feces as a sample, and the kit also contains reagents for extracting total DNA from the feces sample.

3. The kit for gastric cancer screening according to claim 1, characterized in that: The primer set for amplifying the single nucleotide site s1625711 of Helicobacter pylori includes F 1625711 and R 1625711 , F 1625711 :5’-CAAGCCTATAGCGATCGATGAA-3’; R 1625711 :5’-TCAATTCTGCACGCCTTTTG-3’; The primer set for amplifying the single nucleotide site s407233 of Helicobacter pylori includes F 407233 and R 407233 , F 407233 :5’-GGTTATTTAAGGTAGTTGGATGCG-3’; R 407233 :5'-AATAAAAGGGCTTTGCCCCCATTA-3': The probe used to specifically detect the wild type of Helicobacter pylori locus s1625711 is as follows: F 1625711w : 5'-CGCTTTGATGGAAG-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence; The probe used to specifically detect the mutant genotype of Helicobacter pylori locus s1625711 is as follows: F 1625711m： 5'-ACGCTTTAGTGGAAG-3', with HEX modification at the 5' end and mgb and NFQ modifications at the 3' end; Probe for specific detection of wild-type Helicobacter pylori locus s407233: F 407233w : 5'-CTTTTAAAAAAGCATGA-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence; Probe for specific detection of Helicobacter pylori locus s407233 mutation genotype: F 407233m： 5'-TTAAAAGAGCATGATGA-3', wherein the 5' end has HEX modification, and the 3' end has mgb and NFQ modifications in sequence.

4. The kit for gastric cancer screening according to claim 1, characterized in that: It includes a PCR amplification reaction system and a PCR product purification system.

5. A kit for evaluating the prognosis or judging the recurrence and metastasis of gastric cancer after treatment, characterized in that: The kit contains: a primer set for amplifying the single nucleotide sites s1625711 and s407233 of Helicobacter pylori, and a probe for specifically detecting the wild type and mutant genotypes of the single nucleotide sites s1625711 and s407233 of Helicobacter pylori; Among them, the s1625711 site is located in the Helicobacter pylori tRNA ligase gene, and the mutation of the s1625711 site causes the methionine at position 744 to become valine; the s407233 site is located on the gene HP0396, and the mutation of the s407233 site causes the lysine at position 10 to become glutamate.

6. The kit for prognosis assessment or recurrence and metastasis judgment after gastric cancer treatment according to claim 5, characterized in that: The kit is a kit using in vitro feces as a sample, and the kit also contains reagents for extracting total DNA from the feces sample.

7. The kit for prognosis assessment or recurrence and metastasis judgment after gastric cancer treatment according to claim 5, characterized in that: The primer set for amplifying the single nucleotide site s1625711 of Helicobacter pylori includes F 1625711 and R 1625711 , F 1625711 :5’-CAAGCCTATAGCGATCGATGAA-3’; R 1625711 :5’-TCAATTCTGCACGCCTTTTG-3’; The primer set for amplifying the single nucleotide site s407233 of Helicobacter pylori includes F 407233 and R 407233 , F 407233 :5’-GGTTATTTAAGGTAGTTGGATGCG-3’; R 407233 :5'-AATAAAAGGGCTTTGCCCCCATTA-3': The probe used to specifically detect the wild type of Helicobacter pylori locus s1625711 is as follows: F 1625711w : 5'-CGCTTTGATGGAAG-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence; The probe used to specifically detect the mutant genotype of Helicobacter pylori locus s1625711 is as follows: F 1625711m： 5'-ACGCTTTAGTGGAAG-3', with HEX modification at the 5' end and mgb and NFQ modifications at the 3' end; Probe for specific detection of wild-type Helicobacter pylori locus s407233: F 407233w : 5'-CTTTTAAAAAAGCATGA-3', wherein the 5' end is modified with FAM, and the 3' end is modified with mgb and NFQ in sequence; Probe for specific detection of Helicobacter pylori locus s407233 mutation genotype: F 407233m： 5'-TTAAAAGAGCATGATGA-3', wherein the 5' end has HEX modification, and the 3' end has mgb and NFQ modifications in sequence.

8. The kit for prognosis assessment or recurrence and metastasis judgment after gastric cancer treatment according to claim 5, characterized in that: It includes a PCR amplification reaction system and a PCR product purification system.

9. Use of a substance that inhibits single nucleotide site mutation in Helicobacter pylori in the preparation of a vaccine for preventing early gastric cancer, characterized in that: The single nucleotide site is selected from one or both of s1625711 and s407233.

10. A vaccine for preventing early gastric cancer, characterized in that: The vaccine contains a substance that inhibits single nucleotide site mutation of Helicobacter pylori; The single nucleotide site is selected from one or both of s1625711 and s407233.

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