Biomarker and detection reagent for gastric cancer screening

Abstract

The invention relates to the technical field of disease diagnosis, in particular to a biomarker and a detection reagent for gastric cancer screening, the biomarker comprises an intestinal bacteria marker, and intestinal bacteria comprise at least one of Streptococcus pharyngobuccal, Streptococcus constellati, Streptococcus vestibular and Streptococcus parablood. According to the present invention, the gastric cancer risk screening is performed through the joint detection and analysis of the Streptococcus pharyngeal, the Streptococcus constellatus, the Streptococcus vestibulis, the Streptococcus parablood, the Streptococcus vestibulis, and the Streptococcus parablood in the excrement, such that the gastric cancer risk screening can be performed; the invention plays an important role in improving the early diagnosis rate of gastric cancer and reducing the death rate.

Description

Technical Field

[0001] The present invention relates to the technical field of disease diagnosis, and in particular to a biomarker and a detection reagent for gastric cancer screening. Background Art

[0002] Gastric cancer is a malignant tumor that originates from the gastric mucosal epithelium. Its morbidity and mortality rates are both high worldwide, especially in my country, where it ranks first among digestive tract tumors. As a high-incidence area for gastric cancer, China accounts for about 42% of the global total number of new cases each year, a proportion that significantly exceeds the world average. What is more worrying is that its incidence has shown a slow but sustained growth trend in recent years. Globally, gastric cancer is the second most deadly cancer after lung cancer, claiming nearly 800,000 lives each year. In my country, the mortality rate of gastric cancer ranks third among causes of death from malignant tumors, second only to lung cancer and liver cancer, posing a major threat to social health.

[0003] Gastric cancer screening methods include serological examination, barium meal radiography and gastroscopy. Among them, the sensitivity and specificity of serological examination are low, and the diagnostic accuracy of barium meal radiography is relatively low, generally around 30%. Gastroscopy is one of the important means of diagnosing gastric cancer, with the advantages of being clear and intuitive, and being able to detect whether the gastrointestinal tract is diseased or cancerous. Through gastroscopy, doctors can directly observe the state of the gastric mucosa, find suspicious lesions and perform biopsies, thereby confirming or excluding gastric cancer. Therefore, gastroscopy plays a vital role in the early detection and diagnosis of gastric cancer. Gastroscopy requires the insertion of a gastroscope tube through the mouth, which causes a certain degree of discomfort to the patient and may even cause nausea, vomiting and other reactions. This invasiveness makes some patients fear and resist gastroscopy, which in turn affects the popularity and timeliness of screening. Therefore, it is of great significance to develop a non-invasive gastric cancer screening test kit with high diagnostic performance.

[0004] Under the pathological conditions of gastric diseases such as gastric cancer and chronic atrophic gastritis, the pH value of the gastric environment increases abnormally. This change provides living conditions for the oral flora that enter the stomach with food, allowing them to colonize and survive in the stomach. Some of these colonized bacteria will fall off and be excreted from the body with feces, resulting in a significant increase in the content of these specific bacteria in the feces. Zhou Chengbei et al. found that Streptococcus anginosus and Streptococcus constellatus in feces may become non-invasive biomarkers for early warning of gastric cancer and its precancerous lesions (PMID: 35167866). In view of this phenomenon, accurate detection of changes in the content of these bacteria in feces can be used as a non-invasive and potential auxiliary means of gastric cancer screening. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a biomarker for gastric cancer screening to solve the problems in the prior art. At the same time, the present invention will also provide a detection reagent for gastric cancer screening.

[0006] To achieve the above-mentioned purpose and other related purposes, the purpose of the present invention is to provide an intestinal bacterial biomarker for gastric cancer screening, wherein the biomarker includes an intestinal bacterial marker, and the intestinal bacteria include at least one of Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibulus, and Streptococcus parasanguinis.

[0007] In one embodiment of the present invention, the biomarkers include intestinal bacterial markers, and the intestinal bacterial markers include pharyngeal and cheek Streptococcus markers, constitutive Streptococcus markers, vestibular Streptococcus markers and parasanguinis Streptococcus markers.

[0008] In one embodiment of the present invention, the pharyngeal cheek Streptococcus marker comprises any one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.3, or a sequence having an identity of not less than 90% with SEQ ID NO.1 to SEQ ID NO.3;

[0009] The Streptococcus constellatus marker includes any one or more nucleotide sequences of SEQ ID NO.4 to SEQ ID NO.6, or a sequence having an identity of not less than 90% with SEQ ID NO.4 to SEQ ID NO.6;

[0010] The vestibular streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.7 to SEQ ID NO.9, or a sequence having an identity of not less than 90% with SEQ ID NO.7 to SEQ ID NO.9;

[0011] The Streptococcus parasanguinis marker includes any one or more nucleotide sequences of SEQ ID NO.10 to SEQ ID NO.12, or a sequence having an identity of not less than 90% with SEQ ID NO.10 to SEQ ID NO.12.

[0012] In one embodiment of the present invention, the pharyngeal cheek Streptococcus marker comprises any one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.3, or a sequence having an identity of not less than 90% with SEQ ID NO.1 to SEQ ID NO.3;

[0013] The Streptococcus constellatus marker includes any one or more nucleotide sequences of SEQ ID NO.4 to SEQ ID NO.6, or a sequence having an identity of not less than 95% with SEQ ID NO.4 to SEQ ID NO.6;

[0014] The vestibular streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.7 to SEQ ID NO.9, or a sequence having an identity of not less than 95% with SEQ ID NO.7 to SEQ ID NO.9;

[0015] The Streptococcus parasanguinis marker includes any one or more nucleotide sequences of SEQ ID NO.10 to SEQ ID NO.12, or a sequence having an identity of not less than 95% with SEQ ID NO.10 to SEQ ID NO.12.

[0016] In one embodiment of the present invention, the forward primer of the Streptococcus pharyngii is SEQ ID NO.1, or a sequence having an identity of not less than 95% with SEQ ID NO.1;

[0017] The reverse primer of the buccal streptococcus is SEQ ID NO.2, or a sequence having an identity of not less than 95% with SEQ ID NO.2;

[0018] The probe of Streptococcus pharyngii is SEQ ID NO.3, or a sequence having an identity of not less than 95% with SEQ ID NO.3.

[0019] In one embodiment of the present invention, the forward primer of Streptococcus constellatus is SEQ ID NO.4, or a sequence having an identity of not less than 95% with SEQ ID NO.4;

[0020] The reverse primer of the Streptococcus constellatus is SEQ ID NO.5, or a sequence having an identity of not less than 95% with SEQ ID NO.5;

[0021] The probe of Streptococcus constellatus is SEQ ID NO.6, or a sequence having an identity of not less than 95% with SEQ ID NO.6.

[0022] In one embodiment of the present invention, the forward primer of Streptococcus vestibulei is SEQ ID NO.7, or a sequence having an identity of not less than 95% with SEQ ID NO.7;

[0023] The reverse primer of Streptococcus vestibulei is SEQ ID NO.8, or a sequence having an identity of not less than 95% with SEQ ID NO.8;

[0024] The probe of Streptococcus vestibule is SEQ ID NO.9, or a sequence having an identity of not less than 95% with SEQ ID NO.9.

[0025] In one embodiment of the present invention, the forward primer of Streptococcus parasanguinis is SEQ ID NO.10, or a sequence having an identity of not less than 95% with SEQ ID NO.10;

[0026] The reverse primer of Streptococcus vestibulei is SEQ ID NO.11, or a sequence having an identity of not less than 95% with SEQ ID NO.11;

[0027] The probe of Streptococcus parasanguinis is SEQ ID NO.12, or a sequence having an identity of not less than 95% with SEQ ID NO.12.

[0028] In one embodiment of the present invention, the 5' end of the probe for Streptococcus pharyngii is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein;

[0029] The 5' end of the probe for Streptococcus constellatus is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein;

[0030] The 5' end of the vestibular streptococcus probe is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein;

[0031] The 5' end of the Streptococcus parasanguinis probe is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein;

[0032] The 3' end of the probe for Streptococcus pharyngii is labeled with BHQ;

[0033] The 3' end of the probe for Streptococcus constellatus is labeled with BHQ;

[0034] The 3' end of the S. vestibularis probe is labeled with BHQ;

[0035] The 3' end of the Streptococcus parasanguinis probe is labeled with BHQ.

[0036] The second aspect of the present invention provides a detection reagent for gastric cancer screening, wherein the detection reagent comprises the above-mentioned biomarker.

[0037] As described above, the biomarker and detection reagent for gastric cancer screening of the present invention have the following beneficial effects: The present invention provides a non-invasive gastric cancer screening detection method and kit, and performs gastric cancer risk screening by jointly detecting and analyzing pharyngeal buccal streptococci and constellation streptococci, vestibular streptococci (Streptococcus vestibularis), and parasanguinis streptococci (Streptococcus parasanguinis) in feces. The present invention plays an important role in improving the early diagnosis rate of gastric cancer and reducing the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The intestinal flora was analyzed in 50 cases of gastric cancer and 50 cases of non-gastric cancer.

[0039] Figure 2 This is the ROC curve for nucleic acid detection of pharyngeal and cheek Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis in stool samples of 517 gastric cancer patients and 867 non-gastric cancer patients.

[0040] Figure 3 This is the ROC curve of the combined analysis of nucleic acid detection of buccal Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis in feces of independent samples. DETAILED DESCRIPTION

[0041] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0042] Example 1

[0043] Analysis of intestinal flora by 16S rDNA sequencing in gastric cancer

[0044] Fecal DNA extraction

[0045] 16S sequencing was performed on stool samples from 50 gastric cancer patients and 50 non-gastric cancer individuals. Fecal DNA was extracted using the commercial HiPure Stool DNA Mini Kit (MeiGen Biotech) according to the kit instructions.

[0046] First round of PCR

[0047] The corresponding 16S rDNA primers were used according to the selection of the sequencing region. In this example, primers 343F (TACGGRAGGCAGCAG) and 798R (AGGGTATCTAATCCT) were selected. The amplification kit used commercial Tks Gflex DNA Polymerase (TaKaRa) for PCR amplification, and the amplification system was shown in the following table. The PCR amplification program was as follows: 94°C for 1 minute, 1 cycle; 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 20 seconds, 26 cycles; 72°C for 5 minutes, 1 cycle; 4°C hold.

[0048]

[0049]

[0050] Take 5 μl of PCR product and perform electrophoresis on it using a 2% gel concentration to detect whether there are bands and whether the bands are single. After the test is qualified, use commercial PCR product purification magnetic beads to purify it, and use Nanodrop to measure the concentration of the purified PCR product.

[0051] Second round of PCR

[0052] The purified PCR product was used as a second-round PCR template and a second-round PCR amplification was performed. The PCR amplification program was as follows: 94°C for 1 minute, 1 cycle; 98°C for 10 seconds, 56°C for 30 seconds, 72°C for 20 seconds, 7 cycles; 72°C for 5 minutes, 1 cycle; 4°C hold.

[0053]

[0054] The second round of PCR products were tested again by electrophoresis to check whether there were bands and whether the bands were single. After the test was qualified, magnetic beads were used for purification, and the PCR products were quantified by Qubit after purification. Equal amounts of samples were mixed according to the concentration of PCR products and sequenced.

[0055] Data analysis

[0056] After the data is downloaded, the raw data is first cut out of the primer sequence in the raw data sequence using the cutadapt software. Then, DADA2 is used to perform quality control analysis such as quality filtering, noise reduction, splicing, and chimera removal on the qualified double-end raw data in the previous step according to the default parameters of QIIME2 to obtain representative sequences and ASV abundance tables. After selecting the representative sequences of each ASV using the QIIME2 software package, all representative sequences are compared and annotated with the Silva and Greengenes databases. The species comparison annotation is analyzed using the default parameters of the q2-feature-classifier software. LEfSe analysis reveals the composition of species with differences in two or more biological communities.

[0057] Figure 1 The intestinal flora of 50 gastric cancer patients and 50 non-gastric cancer patients were analyzed. A: LDA score of different species. B: The Shannon Index of gastric cancer group and non-gastric cancer group was significantly different (P<0.001). LEfSe data analysis showed that there were significant differences in the composition of intestinal flora between gastric cancer and non-gastric cancer. A total of 26 bacteria with different abundance were screened out, see Figure 1 .

[0058] The abundance of 4 bacteria (Agathobaculum butyriciproducens, Mediterraneibacter_A_155507lactaris, Eubacterium_G ventriosum, Faecalibacillus intestinalis) decreased in gastric cancer patients, while the abundance of 22 bacteria (including Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibularis, Streptococcus parasanguinis, etc.) increased significantly in gastric cancer patients. In addition, the Shannon Index of intestinal flora in gastric cancer patients was also significantly reduced. Because Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis are common bacteria in the oral cavity, and can colonize in the stomach during gastric disease, the abundance of these four bacteria in the feces of gastric cancer patients is increased, so these four bacteria are more ideal biomarkers for gastric cancer.

[0059] In addition, Table 3 shows the ROC curve analysis data of 16S rDNA sequencing of Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis. The ROC curve (Receiver Operating Characteristic curve) analysis shows that the areas under the ROC curves (Area Under Curv, AUC) of Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis are 0.810, 0.775, 0.651, and 0.786, respectively (Table 3). These results preliminarily indicate that fecal Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis are potential biomarkers for gastric cancer screening.

[0060]

[0061] Example 2

[0062] Nucleic acid detection of fecal streptococci, streptococci, vestibular streptococci, and streptococci parasanguinis

[0063] 1. Primers and probes

[0064] Because the content of shed human cells in feces is very low, while the content of Escherichia coli in feces is abundant (10 per gram of feces). 6 -10 8 In this example, Escherichia coli was selected as the internal standard for PCR detection.

[0065] Primer3 and BLAST software were used to design specific primers and probes for Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibules, Streptococcus parasanguis and Escherichia coli. The primer and probe sequences are shown in Table 4. Escherichia coli was used as the internal standard for PCR amplification to monitor the entire process of the detection system. If each target (Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibules, Streptococcus parasanguis) and internal standard (Escherichia coli) were combined into a single tube test, the 5' end of the target and internal standard probes must select different bands of fluorescein.

[0066]

[0067] 2. Experimental testing process

[0068] 2.1 DNA extraction

[0069] Fecal samples from 517 gastric cancer patients (experimental group) and 867 non-gastric cancer patients (control group) were tested for Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis. Fecal DNA was extracted using the commercial HiPureStool DNA Mini Kit (MeiGen Biotech) according to the instructions of the kit.

[0070] Buccal Streptococcus, Constellation Streptococcus, Vestibular Streptococcus, and Parasanguinis Streptococcus were cultured according to the culture conditions in Table 5. The strain content was detected using a digital turbidity meter. The strain DNA was obtained using the commercial HiPure Universal DNA Kit (Meiji Biotechnology) and the operation was carried out according to the instructions of the kit.

[0071]

[0072] 2.2 PCR amplification

[0073] Single-tube single-target detection uses the commercial PCR kit AceQ Universal U+Probe Master MixV2 (NoviZan), that is, each sample and each bacterium to be detected is in a separate tube. If the DNA template is too low, it is concentrated to increase the detection rate.

[0074] Escherichia coli is used as the internal standard for PCR amplification, and the reaction system is shown in Table 6. For weak positive quality control of Streptococcus pharyngeus and cheek, Streptococcus constellatus, Streptococcus vestibule, and Streptococcus parasanguinis, 1000-1500 strain DNA or artificially synthesized plasmid (concentration 1000-1500 copies) can be used. The DNA content of 1000-1500 strains is calculated based on the extracted DNA content, sample extraction volume, and the number of bacteria measured by a digital turbidity meter. For negative quality control, physiological saline is used.

[0075]

[0076] The PCR amplification program was as follows: 37°C for 2 min, 1 cycle; 95°C for 5 min, 1 cycle; 95°C for 10 s, 60°C

[0077] 40 seconds, 42 cycles.

[0078] 3. Test results

[0079] In this embodiment, each batch of tests simultaneously tests negative and positive control products. The positive control product Ct is ≤37.0, and the negative control product Ct>40.0, which proves that the entire test process is effective. If any control product is out of control, it means that the test of this batch is invalid, and the cause needs to be investigated and retested. If the internal standard Ct of Escherichia coli is ≤35.0, the test result is valid. If it is >35.0, the cause needs to be investigated and retested.

[0080] In this embodiment, a Ct value of ≤37.0 for nucleic acid detection of Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibules, and Streptococcus parasanguinis is positive, while a Ct value of >37.0 is negative.

[0081] Among the 517 gastric cancer samples, 477 cases of Streptococcus pharyngii, 491 cases of Streptococcus constellatus, 469 cases of Streptococcus vestibules, and 502 cases of Streptococcus parasanguinis were detected, while in the control group, 80 cases of Streptococcus pharyngii, 110 cases of Streptococcus constellatus, 113 cases of Streptococcus vestibules, and 104 cases of Streptococcus parasanguinis were detected, which was statistically significant (P<0.001, Table 7). The Kappa values ​​of Streptococcus pharyngii, Streptococcus constellatus, Streptococcus vestibules, and Streptococcus parasanguinis were 0.818, 0.797, 0.730, and 0.822, respectively, and the sensitivity was higher than 90%. The specificity of Streptococcus pharyngii was 90.77%, and the other three bacteria were slightly lower than 90%.

[0082]

[0083] Figure 2 The ROC curves for nucleic acid detection of pharyngeal cheek Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis in stool samples of 517 gastric cancer patients and 867 non-gastric cancer patients. ROC curve analysis showed that the AUCs of pharyngeal cheek Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis were 0.915, 0.911, 0.877, and 0.926, respectively (see Appendix). Figure 2 ).

[0084] These results indicate that nucleic acid testing for fecal pharyngeal buccal Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis has good diagnostic performance.

[0085] Example 3

[0086] Combined analysis of fecal pharyngeal and cheek streptococci, constellation streptococci, vestibular streptococci, and parasanguinis streptococci nucleic acid detection

[0087] The nucleic acid test results of Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibules, and Streptococcus parasanguinis in Example 2 were further analyzed for these four bacteria. As shown in Table 8, among the 517 gastric cancer samples, 501 were positive and 16 were negative, while in the control group, the results were 68 and 799, respectively, which was statistically significant (P<0.001).

[0088] The Kappa value of the combined analysis of the four bacteria is 0.873, the sensitivity is 96.91%, the specificity is 92.16%, and the AUC value is 0.985, which has very good diagnostic performance and is a powerful detection method for gastric cancer screening. After analysis, the test result calculation formula obtained in this embodiment is: S value = 0.288 × Ct value (pharyngeal and cheek Streptococcus) + 0.273 × Ct value (constellation Streptococcus) + 0.193 × Ct value (vestibular Streptococcus) + 0.338 × Ct value (parasanguinis). Among them, S value ≤ 1.57197 is a positive result, and S value > 1.57197 is a negative result.

[0089]

[0090] In addition, the four bacteria combined analysis was further performed for stratified analysis. The results showed (Table 9) that the specificity of the four bacteria combined analysis for other gastric diseases was: gastritis 91.18%, gastric ulcer 93.33%, gastric polyp 96.51%, intestinal metaplasia 92.73%, gastric lymphoma 96.08%. The specificity of the four bacteria combined analysis for other types of tumor samples was: esophageal cancer 88.72%, colorectal cancer 91.40%, pancreatic cancer 89.88%, liver cancer 94.59%, lung cancer 93.18%, and other tumors 92.96%.

[0091] These results suggest that combined analysis of the four bacteria has good diagnostic performance for gastric cancer.

[0092]

[0093]

[0094] Example 4

[0095] Independent sample verification of fecal pharyngeal and cheek streptococci, constellation streptococci, vestibular streptococci, and parasanguinis streptococci nucleic acid detection

[0096] The diagnostic performance of fecal nucleic acid detection of Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibules, and Streptococcus parasanguinis for gastric cancer screening was validated in stool samples from 312 gastric cancer and 387 non-gastric cancer individuals.

[0097] According to the detection method of Example 2, the fecal sample was tested for nucleic acid of Streptococcus pharyngeus, Streptococcus constellatus, Streptococcus vestibules, and Streptococcus parasanguinis; and according to the calculation formula of Example 3 for the joint analysis of four bacteria, data processing was performed.

[0098] Figure 3 The ROC curve of the combined analysis of nucleic acid detection of pharyngeal and cheek Streptococcus, constellation Streptococcus, vestibular Streptococcus, and parasanguinis in stool samples of 312 independent gastric cancer and 387 non-gastric cancer individuals. The results of the combined analysis of the four bacteria showed that among the 312 gastric cancer samples, 293 were positive and 19 were negative, while in the control group, they were 18 and 369, respectively, which was statistically significant (P<0.001). The Kappa value of the combined analysis of the four bacteria was 0.893, the sensitivity was 93.91%, the specificity was 95.35%, and the AUC value was 0.946, which has very good diagnostic performance and is a powerful detection method for gastric cancer screening.

[0099] In summary, the present invention verifies that the biomarkers of fecal pharyngeal buccal streptococci, constellation streptococci, vestibular streptococci, and parasanguinis streptococci can be used to screen for gastric cancer, have very good diagnostic performance, and are powerful detection means for gastric cancer screening. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0100] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A biomarker for gastric cancer screening, characterized in that: The biomarkers include intestinal bacteria markers, and the intestinal bacteria include at least one of Streptococcus pharyngeus and cheek, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis.

2. A biomarker for gastric cancer screening according to claim 1, characterized in that: The intestinal bacteria markers include buccal streptococcus markers, constellation streptococcus markers, vestibular streptococcus markers and parasanguinis streptococcus markers.

3. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The pharyngeal cheek streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.3, or a sequence having an identity of not less than 90% with SEQ ID NO.1 to SEQ ID NO.3; The Streptococcus constellatus marker includes any one or more nucleotide sequences of SEQ ID NO.4 to SEQ ID NO.6, or a sequence having an identity of not less than 90% with SEQ ID NO.4 to SEQ ID NO.6; The vestibular streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.7 to SEQ ID NO.9, or a sequence having an identity of not less than 90% with SEQ ID NO.7 to SEQ ID NO.9; The Streptococcus parasanguinis marker includes any one or more nucleotide sequences of SEQ ID NO.10 to SEQ ID NO.12, or a sequence having an identity of not less than 90% with SEQ ID NO.10 to SEQ ID NO.

12.

4. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The pharyngeal cheek streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.3, or a sequence having an identity of not less than 90% with SEQ ID NO.1 to SEQ ID NO.3; The Streptococcus constellatus marker includes any one or more nucleotide sequences of SEQ ID NO.4 to SEQ ID NO.6, or a sequence having an identity of not less than 95% with SEQ ID NO.4 to SEQ ID NO.6; The vestibular streptococcus marker includes any one or more nucleotide sequences of SEQ ID NO.7 to SEQ ID NO.9, or a sequence having an identity of not less than 95% with SEQ ID NO.7 to SEQ ID NO.9; The Streptococcus parasanguinis marker includes any one or more nucleotide sequences of SEQ ID NO.10 to SEQ ID NO.12, or a sequence having an identity of not less than 95% with SEQ ID NO.10 to SEQ ID NO.

12.

5. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The forward primer of the buccal streptococcus is SEQ ID NO.1, or a sequence having an identity of not less than 95% with SEQ ID NO.1; The reverse primer of the buccal streptococcus is SEQ ID NO.2, or a sequence having an identity of not less than 95% with SEQ ID NO.2; The probe of Streptococcus pharyngii is SEQ ID NO.3, or a sequence having an identity of not less than 95% with SEQ ID NO.

3.

6. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The forward primer of Streptococcus constellatus is SEQ ID NO.4, or a sequence having an identity of not less than 95% with SEQ ID NO.4; The reverse primer of the Streptococcus constellatus is SEQ ID NO.5, or a sequence having an identity of not less than 95% with SEQ ID NO.5; The probe of Streptococcus constellatus is SEQ ID NO.6, or a sequence having an identity of not less than 95% with SEQ ID NO.

6.

7. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The forward primer of Streptococcus vestibulei is SEQ ID NO.7, or a sequence having an identity of not less than 95% with SEQ ID NO.7; The reverse primer of Streptococcus vestibulei is SEQ ID NO.8, or a sequence having an identity of not less than 95% with SEQ ID NO.8; The probe of Streptococcus vestibule is SEQ ID NO.9, or a sequence having an identity of not less than 95% with SEQ ID NO.

9.

8. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The forward primer of Streptococcus parasanguinis is SEQ ID NO.10, or a sequence having an identity of not less than 95% with SEQ ID NO.10; The reverse primer of Streptococcus vestibulei is SEQ ID NO.11, or a sequence having an identity of not less than 95% with SEQ ID NO.11; The probe of Streptococcus parasanguinis is SEQ ID NO.12, or a sequence having an identity of not less than 95% with SEQ ID NO.

12.

9. A biomarker for gastric cancer screening according to claim 1 or 2, characterized in that: The 5' end of the probe for the buccal streptococcus is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein; The 5' end of the probe for Streptococcus constellatus is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein; The 5' end of the vestibular streptococcus probe is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein; The 5' end of the Streptococcus parasanguinis probe is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein; The 3' end of the probe for Streptococcus pharyngii is labeled with BHQ; The 3' end of the probe for Streptococcus constellatus is labeled with BHQ; The 3' end of the S. vestibularis probe is labeled with BHQ; The 3' end of the Streptococcus parasanguinis probe is labeled with BHQ.

10. A detection reagent for gastric cancer screening, characterized in that: The detection reagent comprises the biomarker according to any one of claims 1 to 9.

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