A biomarker and detection reagent for gastric cancer screening

Abstract

The present application relates to the technical field of disease diagnosis, in particular to a biomarker and detection reagent for gastric cancer screening, wherein the biomarker comprises an intestinal bacterial marker, and the intestinal bacteria comprise at least one of Streptococcus anginosus, Streptococcus constellatus, Streptococcus vestibularis and Streptococcus parasanguinis. The present application performs gastric cancer risk screening by jointly detecting and analyzing Streptococcus anginosus, Streptococcus constellatus, Streptococcus vestibularis and Streptococcus parasanguinis in feces. The present application plays an important role in improving the early diagnosis rate and reducing the mortality rate of gastric cancer.

Description

Technical Field

[0001] This invention relates to the field of disease diagnosis technology, and in particular to a biomarker and detection reagent for gastric cancer screening. Background Technology

[0002] Stomach cancer is a malignant tumor originating from the gastric mucosal epithelium. Its incidence and mortality rates are among the highest globally, especially in my country, where it ranks first among digestive tract cancers. As a high-incidence region for stomach cancer, China accounts for approximately 42% of new cases globally each year, a proportion significantly exceeding the world average. More worryingly, its incidence rate has shown a slow but continuous upward trend in recent years. Globally, stomach cancer is the second leading cause of cancer death after lung cancer, claiming nearly 800,000 lives annually. In my country, however, stomach cancer ranks third among malignant tumor causes of death, after lung and liver cancer, posing a significant threat to public health.

[0003] Gastric cancer screening methods include serological testing, barium meal radiography, and gastroscopy. Among these, serological testing has relatively low sensitivity and specificity, while barium meal radiography has a relatively low diagnostic accuracy, generally around 30%. Gastroscopy is a crucial method for diagnosing gastric cancer, offering advantages such as clear visualization and the ability to detect gastrointestinal lesions or cancerous changes. Through gastroscopy, doctors can directly observe the condition of the gastric mucosa, identify suspicious lesions, and perform biopsies, thereby confirming or ruling out gastric cancer. Therefore, gastroscopy plays a vital role in the early detection and diagnosis of gastric cancer. However, gastroscopy requires the insertion of a gastroscope tube through the mouth, causing discomfort to patients and potentially triggering nausea and vomiting. This invasiveness leads some patients to fear and resist gastroscopy, thus affecting the coverage and timeliness of screening. Therefore, the development of non-invasive, high-diagnostic-performance gastric cancer screening kits is of great significance.

[0004] In the pathological states of gastric diseases such as gastric cancer and chronic atrophic gastritis, the pH value of the gastric environment abnormally increases. This change provides conditions for the survival of oral flora that enter the stomach with food, allowing them to colonize and survive in the stomach. Some of these colonized bacteria detach and are excreted with feces, leading to a significant increase in the content of these specific bacteria in feces. Zhou Chengbei et al. found that *Streptococcus anginosus* and *Streptococcus constellatus* in feces may serve as non-invasive biomarkers for early warning of gastric cancer and its precancerous lesions (PMID: 35167866). Given this phenomenon, accurately detecting changes in the content of these bacteria in feces could serve as a non-invasive, potential adjunctive method for gastric cancer screening. Summary of the Invention

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

[0006] To achieve the above and other related objectives, the present invention aims to provide an intestinal bacterial biomarker for gastric cancer screening, wherein the biomarker includes intestinal bacterial biomarkers, and the intestinal bacteria include at least one of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus.

[0007] In one embodiment of the present invention, the biomarkers include intestinal bacterial markers, which include buccal streptococcus markers, constellation streptococcus markers, vestibular streptococcus markers, and parablood streptococcus markers.

[0008] In one embodiment of the present invention, the buccal streptococcus marker includes any one or more nucleotide sequences from SEQ ID NO.1 to SEQ ID NO.3, or sequences having not less than 90% identity with SEQ ID NO.1 to SEQ ID NO.3;

[0009] The constellation streptococcal marker includes any one or more nucleotide sequences from SEQ ID NO.4 to SEQ ID NO.6, or sequences having at least 90% identity with SEQ ID NO.4 to SEQ ID NO.6;

[0010] The vestibular streptococcal marker includes any one or more nucleotide sequences from SEQ ID NO.7 to SEQ ID NO.9, or sequences having at least 90% identity with SEQ ID NO.7 to SEQ ID NO.9;

[0011] The parablood streptococcal markers include any one or more nucleotide sequences from SEQ ID NO.10 to SEQ ID NO.12, or sequences having at least 90% identity with SEQ ID NO.10 to SEQ ID NO.12.

[0012] In one embodiment of the present invention, the buccal streptococcus marker includes any one or more nucleotide sequences from SEQ ID NO.1 to SEQ ID NO.3, or sequences having not less than 90% identity with SEQ ID NO.1 to SEQ ID NO.3;

[0013] The constellation streptococcal markers include any one or more nucleotide sequences from SEQ ID NO.4 to SEQ ID NO.6, or sequences having at least 95% identity with SEQ ID NO.4 to SEQ ID NO.6;

[0014] The vestibular streptococcal marker includes any one or more nucleotide sequences from SEQ ID NO.7 to SEQ ID NO.9, or sequences having at least 95% identity with SEQ ID NO.7 to SEQ ID NO.9;

[0015] The parablood streptococcal markers include any one or more nucleotide sequences from SEQ ID NO.10 to SEQ ID NO.12, or sequences having at least 95% identity with SEQ ID NO.10 to SEQ ID NO.12.

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

[0017] The reverse primer for the buccal streptococcus is SEQ ID NO.2, or a sequence having at least 95% identity with SEQ ID NO.2;

[0018] The probe for the buccal streptococcus is SEQ ID NO.3, or a sequence having at least 95% identity with SEQ ID NO.3.

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

[0020] The reverse primer for the Streptococcus constellations is SEQ ID NO.5, or a sequence having at least 95% identity with SEQ ID NO.5;

[0021] The probe for the *Streptococcus constellans* is SEQ ID NO. 6, or a sequence having at least 95% identity with SEQ ID NO. 6.

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

[0023] The reverse primer for the vestibular streptococcus is SEQ ID NO.8, or a sequence having at least 95% identity with SEQ ID NO.8;

[0024] The probe for the vestibular streptococcus is SEQ ID NO.9, or a sequence having at least 95% identity with SEQ ID NO.9.

[0025] In one embodiment of the present invention, the forward primer for the parahaemolytic streptococcus is SEQ ID NO.10, or a sequence having at least 95% identity with SEQ ID NO.10;

[0026] The reverse primer for the vestibular streptococcus is SEQ ID NO.11, or a sequence having at least 95% identity with SEQ ID NO.11;

[0027] The probe for the parablood streptococcus is SEQ ID NO.12, or a sequence having at least 95% identity with SEQ ID NO.12.

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

[0030] The 5' end of the probe for *Streptococcus vestibulus* is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein, or CY system fluorescein.

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

[0032] The probe for Streptococcus bag is labeled with BHQ at its 3' end;

[0033] The probe of the constellation Streptococcus is labeled with BHQ at its 3' end;

[0034] The 3' end of the probe for Streptococcus vestibulus is labeled with BHQ;

[0035] The probe for *Streptococcus parahaemolyticus* is labeled with BHQ at its 3' end.

[0036] In a second aspect, the present invention provides a detection reagent for gastric cancer screening, the detection reagent comprising the above-mentioned biomarkers.

[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, which performs gastric cancer risk screening by jointly detecting and analyzing Streptococcus buccali, Streptococcus constellans, Streptococcus vestibularis, and 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. Attached Figure Description

[0038] Figure 1 The gut microbiota of 50 patients with gastric cancer and 50 patients without gastric cancer were analyzed.

[0039] Figure 2 ROC curves for nucleic acid detection of Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus in stool samples from 517 patients with gastric cancer and 867 patients without gastric cancer.

[0040] Figure 3 ROC curves for combined analysis of nucleic acid detection of Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus in independent fecal samples. Detailed Implementation

[0041] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0042] Example 1

[0043] 16S rDNA sequencing and gut microbiota analysis for gastric cancer

[0044] Fecal DNA extraction

[0045] 16S sequencing was performed on stool samples from 50 patients with gastric cancer and 50 individuals without gastric cancer. Fecal DNA extraction was performed using the commercially available HiPure Stool DNA Mini Kit (Meiji Biotechnology), following the kit's instructions.

[0046] First round of PCR

[0047] The appropriate 16S rDNA primers were used based on the selected sequencing region. In this example, primers 343F (TACGGRAGGCAGCAG) and 798R (AGGGTATCTAATCCT) were selected. PCR amplification was performed using the commercially available Tks Gflex DNA Polymerase (TaKaRa). The amplification system is shown in the table below. The PCR amplification program was as follows: 94℃ for 1 minute, 1 cycle; 98℃ for 10 seconds, 56℃ for 30 seconds, 72℃ for 20 seconds, 26 cycles; 72℃ for 5 minutes, 1 cycle; 4℃ hold.

[0048]

[0049]

[0050] Take 5 μl of PCR product and perform electrophoresis on a 2% gel to detect the presence and uniformity of bands. After passing the detection, purify the PCR product using commercially available PCR product purification magnetic beads, and determine the concentration of the purified PCR product using Nanodrop.

[0051] Second round of PCR

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

[0053]

[0054] The second round of PCR products was analyzed by electrophoresis to check for the presence and uniformity of bands. After passing the initial analysis, the products were purified using magnetic beads, and then quantified using a Qubit algorithm. Equal volumes of the PCR products were then pooled according to their concentrations and sequenced.

[0055] Data Analysis

[0056] After the data was processed, the raw data was first cut using the cutadapt software to remove primer sequences from the raw data sequences. Then, DADA2 was used to perform quality control analyses on the qualified paired-end raw data from the previous step, including quality filtering, noise reduction, splicing, and chimera removal, according to the default parameters of QIIME2, to obtain representative sequences and an ASV abundance table. Representative sequences for each ASV were selected using the QIIME2 software package, and all representative sequences were aligned and annotated against the Silva and Greengenes databases. Species alignment and annotation were performed using the q2-feature-classifier software with default parameters. LEfSe analysis revealed the composition of different species in two or more biological communities.

[0057] Figure 1 Gut microbiota analysis was performed on 50 patients with gastric cancer and 50 patients without gastric cancer. A: LDA score plot of differentially expressed species. B: Significant difference in Shannon index between the gastric cancer group and the control group (P<0.001). LEfSe data analysis showed significant differences in gut microbiota composition between gastric cancer and non-gastric cancer patients, and 26 bacteria with differential abundance were screened. Figure 1 .

[0058] Four bacteria (Agathobaculum butyriciproducens, Mediturneibacter A155507lactaris, Eubacterium ventriosum, and Faecalibacillus intestinalis) showed decreased abundance in gastric cancer patients, while 22 bacteria (including Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus) showed significantly increased abundance. Furthermore, the Shannon Index of the gut microbiota was also significantly reduced in gastric cancer patients. Since Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus are common bacteria in the oral cavity and can colonize the stomach in cases of gastric disease, and their abundance is increased in the feces of gastric cancer patients, these four bacteria are relatively ideal biomarkers for gastric cancer.

[0059] In addition, Table 3 shows the ROC curve analysis data of 16S rDNA sequencing of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus. ROC curve (Receiver Operating Characteristic curve) analysis showed that the areas under the ROC curves (AUC) for Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus were 0.810, 0.775, 0.651, and 0.786, respectively (Table 3). These results preliminarily suggest that Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus in feces are potential biomarkers for gastric cancer screening.

[0060]

[0061] Example 2

[0062] Nucleic acid detection of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parabloodi in feces

[0063] 1. Primers and probes

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

[0065] Primers and probes specific to *Streptococcus buccali*, *Streptococcus constellans*, *Streptococcus vestibulum*, *Streptococcus parahaemolyticus*, and *Escherichia coli* were designed using Primer3 and BLAST software. Primer and probe sequences are shown in Table 4. *Escherichia coli* was used as an internal control for PCR amplification to monitor the entire detection process. If each target (*Streptococcus buccali*, *Streptococcus constellans*, *Streptococcus vestibulum*, *Streptococcus parahaemolyticus*) and internal control (*Escherichia coli*) were combined into a single tube detection, the 5' ends of the target and internal control probes must be selected with fluorescein at different wavelengths.

[0066]

[0067] 2. Experimental Testing Procedure

[0068] 2.1 DNA extraction

[0069] Fecal samples from 517 patients with gastric cancer (experimental group) and 867 patients without gastric cancer (control group) were tested for Streptococcus buccali, Streptococcus constitutus, Streptococcus vestibulum, and Streptococcus parahaemolyticus. Fecal DNA extraction was performed using the commercially available HiPureStool DNA Mini Kit (Meiji Biotechnology), following the kit's instructions.

[0070] Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus were cultured according to the conditions in Table 5. The bacterial count was detected using a digital turbidimeter, and the bacterial DNA was analyzed using the commercial HiPure Universal DNA Kit (Meiji Biotechnology). The procedure was performed according to the kit's instructions.

[0071]

[0072] 2.2 PCR amplification

[0073] Single-tube, single-target detection uses the commercially available PCR kit AceQ Universal U+Probe Master MixV2 (Novozymes), meaning each sample and each bacterium to be tested is tested in a separate tube. If the DNA template is too low, it is concentrated to improve the detection rate.

[0074] Escherichia coli was used as an internal standard for PCR amplification; the reaction system is shown in Table 6. For weakly positive control samples of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus, 1000-1500 copies of bacterial DNA or a synthetic plasmid (concentration 1000-1500 copies) could be used. The 1000-1500 copies of bacterial DNA were calculated based on the extracted DNA content, sample volume, and the number of bacteria measured by a digital turbidimeter. Physiological saline was used as the negative control.

[0075]

[0076] The PCR amplification program is as follows: 37℃ for 2 minutes, 1 cycle; 95℃ for 5 minutes, 1 cycle; 95℃ for 10 seconds, 60℃

[0077] 40 seconds, 42 cycles.

[0078] 3. Test Results

[0079] In this embodiment, negative and positive quality controls are tested simultaneously for each batch. A positive quality control Ct ≤ 37.0 and a negative quality control Ct > 40.0 indicate the validity of the entire testing procedure. If any quality control fails to meet the control requirements, the batch test is invalid and the cause must be investigated before retesting. An internal standard Ct for Escherichia coli ≤ 35.0 indicates a valid test result; if Ct > 35.0, the cause must be investigated before retesting.

[0080] In this embodiment, a Ct value ≤37.0 for nucleic acid detection of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus is considered positive, while a value >37.0 is considered negative.

[0081] In 517 gastric cancer samples, 477 cases of *Streptococcus buccum*, 491 cases of *Streptococcus constellans*, 469 cases of *Streptococcus vestibulum*, and 502 cases of *Streptococcus parahaemolyticus* were detected. In the control group, 80 cases of *Streptococcus buccum*, 110 cases of *Streptococcus constellans*, 113 cases of *Streptococcus vestibulum*, and 104 cases of *Streptococcus parahaemolyticus* were detected, showing statistically significant differences (P<0.001, Table 7). The Kappa values ​​of *Streptococcus buccum*, *Streptococcus constellans*, *Streptococcus vestibulum*, and *Streptococcus parahaemolyticus* were 0.818, 0.797, 0.730, and 0.822, respectively, with sensitivities all above 90%. The specificity of *Streptococcus buccum* was 90.77%, while the specificity of the other three bacteria was slightly below 90%.

[0082]

[0083] Figure 2 ROC curves were plotted for nucleic acid detection of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus in stool samples from 517 gastric cancer patients and 867 non-gastric cancer patients. ROC curve analysis showed that the AUCs for Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus were 0.915, 0.911, 0.877, and 0.926, respectively (see attached figure). Figure 2 ).

[0084] These results demonstrate that the nucleic acid detection of Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulus, and Streptococcus parabloodi in feces has good diagnostic performance.

[0085] Example 3

[0086] Combined analysis of fecal buccal streptococci, constellation streptococci, vestibular streptococci, and parablood streptococci nucleic acid detection

[0087] The nucleic acid detection results of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus in Example 2 were further analyzed using a combined analysis of these four bacteria. As shown in Table 8, among 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 combined analysis of the four bacteria yielded a Kappa value of 0.873, a sensitivity of 96.91%, a specificity of 92.16%, and an AUC of 0.985, demonstrating excellent diagnostic performance and making it a powerful detection method for gastric cancer screening. In this embodiment, the calculation formula for the detection result is: S value = 0.288 × Ct value (Buccal Streptococcus) + 0.273 × Ct value (Constituent Streptococcus) + 0.193 × Ct value (Vestibular Streptococcus) + 0.338 × Ct value (Parasitic Streptococcus). An S value ≤ 1.57197 indicates a positive result, and an S value > 1.57197 indicates a negative result.

[0089]

[0090] Furthermore, a stratified analysis was performed using a four-bacterial co-analysis. The results (Table 9) show that the specificity of the four-bacterial co-analysis for other gastric diseases was: gastritis 91.18%, gastric ulcer 93.33%, gastric polyps 96.51%, intestinal metaplasia 92.73%, and gastric lymphoma 96.08%. The specificity of the four-bacterial co-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 four bacteria has good diagnostic performance for gastric cancer.

[0092]

[0093]

[0094] Example 4

[0095] Independent sample validation for fecal buccal streptococci, constellation streptococci, vestibular streptococci, and parablood streptococci nucleic acid detection

[0096] The diagnostic performance of fecal streptococci, streptococci, vestibular streptococci, and parablood streptococci nucleic acid detection for gastric cancer screening was validated in stool samples from 312 individuals with gastric cancer and 387 individuals without gastric cancer.

[0097] According to the detection method in Example 2, nucleic acid detection of Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus in fecal samples was performed; data processing was performed according to the calculation formula for the joint analysis of the four bacteria in Example 3.

[0098] Figure 3 The ROC curves for combined nucleic acid analysis of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus in stool samples from 312 independent individuals with gastric cancer and 387 individuals without gastric cancer are shown. The combined analysis of the four bacteria revealed 293 positive and 19 negative results in the 312 gastric cancer samples, compared to 18 and 369 negative results in the control group, respectively (P<0.001). The Kappa value of the combined analysis of the four bacteria was 0.893, with a sensitivity of 93.91%, a specificity of 95.35%, and an AUC of 0.946, demonstrating excellent diagnostic performance and making it a powerful detection method for gastric cancer screening.

[0099] In summary, this invention verifies that fecal biomarkers of Streptococcus buccali, Streptococcus constellans, Streptococcus vestibulum, and Streptococcus parahaemolyticus can be used for gastric cancer screening, exhibiting excellent diagnostic performance and serving as a powerful detection method for gastric cancer screening. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and possesses high industrial application value.

[0100] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A biomarker detection reagent composition for gastric cancer screening, characterized in that, The biomarker detection reagents are respectively biomarker detection reagents for Streptococcus anginosus, Streptococcus constellatus, Streptococcus vestibularis, and Streptococcus parasanguinis. The detection reagent for Streptococcus buccal biomarker is a nucleotide fragment with the sequence shown in SEQ ID NO.1 to SEQ ID NO.3, wherein the forward primer for Streptococcus buccal is SEQ ID NO.1, the reverse primer for Streptococcus buccal is SEQ ID NO.2, and the probe for Streptococcus buccal is SEQ ID NO.3; The Streptococcus constellans marker detection reagent is a nucleotide fragment with the sequence shown in SEQ ID NO.4 to SEQ ID NO.6, wherein the forward primer for Streptococcus constellans is SEQ ID NO.4, the reverse primer for Streptococcus constellans is SEQ ID NO.5, and the probe for Streptococcus constellans is SEQ ID NO.6; The detection reagent for the vestibular streptococcus marker is a nucleotide fragment with the sequence shown in SEQ ID NO.7 to SEQ ID NO.9, wherein the forward primer for vestibular streptococcus is SEQ ID NO.7, the reverse primer for vestibular streptococcus is SEQ ID NO.8, and the probe for vestibular streptococcus is SEQ ID NO.9; The parahaemolyticus marker detection reagent is a nucleotide fragment with the sequence shown in SEQ ID NO.10 to SEQ ID NO.12, wherein the forward primer for parahaemolyticus is SEQ ID NO.10, the reverse primer for parahaemolyticus is SEQ ID NO.11, and the probe for parahaemolyticus is SEQ ID NO.

12.

2. The detection reagent composition according to claim 1, characterized in that, The probe for Streptococcus bag is labeled with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein at its 5' end; The probes for the constellation streptococci are labeled at their 5' ends with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein. The probe for the vestibular streptococcus is labeled at its 5' end with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein or CY system fluorescein; The probe for *Streptococcus parahaemolyticus* is labeled at its 5' end with FAM, HEX, TET, ROX, VIC, NED, Alexa Flour, ATTO series fluorescein, or CY system fluorescein. Furthermore, the 5' end labels of the probes for Streptococcus buccalis, Streptococcus constellans, Streptococcus vestibulae, and Streptococcus parahaemolyticus are all different from each other; The probe for Streptococcus pharynx is labeled with BHQ at its 3' end; The probe of the constellation Streptococcus is labeled with BHQ at its 3' end; The probe for the vestibular streptococcus is labeled with BHQ at its 3' end; The probe for *Streptococcus parahaemolyticus* is labeled with BHQ at its 3' end.

3. The use of the detection reagent composition of claim 1 or 2 in the preparation of a kit for gastric cancer screening.

4. A gastric cancer screening kit, wherein the detection reagent is the detection reagent composition of claim 1 or 2.

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