Application of bacteria as nasopharynx cancer detection, treatment or prognosis marker

Abstract

The invention belongs to the field of biological medicine, and relates to application of bacteria as a marker for detection, treatment or prognosis of nasopharyngeal carcinoma. As a marker, the bacterium disclosed by the invention can be used for prevention, diagnosis, treatment and prognosis intervention of nasopharynx cancer. Through verification, the marker has relatively high diagnosis efficiency and accuracy on diagnosis and prognosis of nasopharynx cancer, an economical, simple and convenient solution is provided for clinic, and a new insight is also provided for accurate individualized treatment of nasopharynx cancer patients.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of bacteria as markers for the detection, treatment or prognosis of nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma is a malignant tumor originating from the nasopharyngeal epithelium. Genetic factors, environmental factors, Epstein-Barr virus infection, and the interaction of the three are the main causes of nasopharyngeal carcinoma. The nasopharynx is connected to the external environment through the mouth and nose, and is colonized by a rich variety of bacteria. They are key members of the local microenvironment and play an important role in the colonization of pathogenic microorganisms, viral infection, and the shaping of the immune microenvironment. Previous studies have revealed the important role of nasopharyngeal flora in respiratory infection-related diseases, and observed that the nasopharyngeal flora is associated with the prognosis and treatment response of nasopharyngeal carcinoma. The resident flora colonized in the nasopharyngeal mucosa plays an important role in maintaining the stability of the local microecology, resisting the colonization of opportunistic pathogens, and regulating the balance of mucosal immunity. It is the cornerstone of maintaining nasopharyngeal health. The imbalance of nasopharyngeal flora may increase the colonization opportunities of other pathogens by upregulating the expression of adhesion-related receptors in the mucosal epithelium and inducing local inflammatory responses, and may promote the infection of respiratory viruses to nasopharyngeal epithelial cells.

[0003] Crosstalk between microbiota in different parts of the body has been found to be associated with a variety of diseases, among which the phenomenon of oral microbiota appearing in distant organs outside the oral cavity has attracted much attention. Species traditionally defined as oral pathogens appear in the feces and lesions of patients with various digestive tract tumors. For example, oral opportunistic pathogens such as Fusobacterium nucleatum are significantly enriched in the feces and cancer tissues of patients with colorectal cancer and high-risk intestinal adenomas. The nasopharynx is the site of nasopharyngeal carcinoma and is anatomically connected to the oral cavity. However, in nasopharyngeal carcinoma, the relationship between the oral and nasopharyngeal microbiota remains unclear.

[0004] In view of the key role of the microbiota in diseases and its interventional characteristics, intervention studies on the microbiota have begun to rise in recent years, and a number of new microbiota therapies based on fecal microbiota transplantation, probiotics / metabiotics supplementation, etc. have entered the clinic. Discovering changes in the nasopharyngeal microbiota associated with nasopharyngeal carcinoma is a prerequisite for the development of microbiota intervention strategies for nasopharyngeal carcinoma, and revealing the impact of changes in the nasopharyngeal microbiota on the tumor microenvironment and Epstein-Barr virus infection is the theoretical basis for intervention research. At present, there is still little research on the above two aspects, which needs to be improved. Summary of the invention

[0005] On the one hand, the present application provides the use of a bacterial detection reagent in preparing a product;

[0006] The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum;

[0007] The product has one or more of the following functions:

[0008] A1. Predict the risk of nasopharyngeal carcinoma;

[0009] A2. Diagnosis or auxiliary diagnosis of nasopharyngeal carcinoma;

[0010] A3. Predict the risk of recurrence of nasopharyngeal carcinoma;

[0011] A4. Predict the risk of nasopharyngeal carcinoma metastasis;

[0012] A5. Predict disease-free survival in patients with nasopharyngeal carcinoma;

[0013] A6. Predicting distant metastasis-free survival in patients with nasopharyngeal carcinoma;

[0014] A7. Predict overall survival of patients with nasopharyngeal carcinoma;

[0015] A8. Prognosis prediction for patients with nasopharyngeal carcinoma.

[0016] In some embodiments, the detection reagent is a primer, a probe, an antisense oligonucleotide, an aptamer, and / or an antibody for detecting bacteria.

[0017] In some embodiments, the detection reagent is a reagent for detecting bacterial abundance; in some embodiments, the reagent for detecting bacterial abundance is a 16S rRNA detection reagent, a qPCR detection reagent, an RNAseq detection reagent, a high-throughput sequencing reagent, or a metagenomic sequencing reagent; in some embodiments, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V1-V9 region; in some embodiments, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V4 region; in some embodiments, the reagent for detecting bacterial abundance is a primer; in some embodiments, the primers are 27F and 1492R primers; in some embodiments, the sequences of the primers are as shown in SEQ ID NOs: 1 and 2.

[0018] In one aspect, the present application provides a reagent for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, comprising a detection reagent for bacteria, wherein the bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

[0019] In some embodiments, the reagents also include extraction reagents for saliva, tissue, nasal mucus, nasal wash fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal wash fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells; in some embodiments, the reagents are kits, test strips and / or chips.

[0020] In one aspect, the present application provides a system for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, comprising:

[0021] Information acquisition module: obtaining the bacterial abundance in the subject's sample;

[0022] Data analysis module: Based on the abundance of bacteria in the subjects' samples, the risk of occurrence, recurrence, and metastasis of nasopharyngeal carcinoma is determined, specifically the probability of developing nasopharyngeal carcinoma, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival;

[0023] Result output module: output evaluation results;

[0024] The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

[0025] In some embodiments, the information acquisition module includes the reagent.

[0026] In some embodiments, the judgment criteria of the data analysis module are: if the bacterial abundance is higher than a threshold, the probability of occurrence, recurrence, metastasis risk, poor prognosis, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival of the subject's nasopharyngeal carcinoma is higher than that of a healthy population;

[0027] If the bacterial abundance is lower than the threshold, the subjects' chances of nasopharyngeal carcinoma development, recurrence, metastasis risk, poor prognosis, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival are lower than those of the general population.

[0028] In one aspect, the present application provides an electronic device for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, the electronic device comprising:

[0029] An acquisition and detection module is configured to acquire a biological sample, detect the biological sample, and obtain the abundance of bacteria in the biological sample;

[0030] a prevention, diagnosis, and / or prognosis module, wherein the prevention, diagnosis, and / or prognosis module outputs a prevention, diagnosis, and / or prognosis result according to a model based on the bacterial abundance;

[0031] The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

[0032] In some embodiments, the subject's sample or biological sample includes: saliva, tissue, nasal mucus, nasal wash fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal wash fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells; in some embodiments, the subject includes healthy people and nasopharyngeal cancer patients.

[0033] In one aspect, the present application provides a method for constructing a model for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, the method comprising:

[0034] Testing the biological sample taken from the subject to obtain a bacterial flora dataset;

[0035] Obtaining the subject's condition of suffering from nasopharyngeal carcinoma, and constructing a first data set using the microbiome data set and the condition of suffering from nasopharyngeal carcinoma;

[0036] Performing multivariate statistics and data mining on the first data set, performing species difference analysis on the bacterial community data set, and obtaining the bacterial species markers associated with the nasopharyngeal carcinoma;

[0037] The biological sample includes nasal mucus, nasal washing fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal washing fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells;

[0038] The subjects include healthy people and nasopharyngeal cancer patients;

[0039] The bacterial strain marker is Prevotella intermedia and / or Fusobacterium nucleatum.

[0040] In one aspect, the present application provides the use of a bacterial inhibitor or an antibacterial drug in preparing a product, wherein the bacteria are Prevotella intermedia and / or Fusobacterium nucleatum;

[0041] The product has one or more of the following functions:

[0042] B1. Anti-EB virus;

[0043] B2. Inhibit the proliferation of nasopharyngeal carcinoma cells;

[0044] B3. Inhibit the migration of nasopharyngeal carcinoma cells.

[0045] In some embodiments, the product is a drug for treating nasopharyngeal carcinoma.

[0046] In one aspect, the present application provides the use of a bacterial inhibitor or an antibacterial drug in resisting Epstein-Barr virus infection, wherein the bacteria are Prevotella intermedia and / or Fusobacterium nucleatum. In some embodiments, the anti-Epstein-Barr virus infection is anti-Epstein-Barr virus infection of nasopharyngeal epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The enriched bacteria in NPC are closely related to the oral ectopic phenomenon; A. Violin plot of the alpha diversity Shannon index of nasopharyngeal and oral flora; B. PCoA plot based on Bray-Curtis distance reflects the distribution of flora structure in the two parts of NPC patients and the control group (the ellipse is 75% CI); C. Bacterial similarity in paired data of oral and nasopharyngeal flora in NPC patients and the control group (based on Bray-Curtis distance); D. Difference test of nasopharyngeal ectopic scores of oral flora in NPC patients and the control group (two-group rank sum test).

[0048] Figure 2 Figure 2. Oral-to-nasopharyngeal microbiota heterotopia phenomenon and microbiota characteristics associated with heterotopia. A. The distribution of oral-to-nasopharyngeal microbiota heterotopia scores between NPC and control groups, evaluated using FEAST and sourcetracker algorithms, and presented using k-means clustering. B. The differential species between high and low microbiota heterotopia groups, calculated based on the ANCOM-BC method, with the model corrected for gender, age, smoking, drinking, and oral and nasopharyngeal diseases, and species with q<0.05 after FDR correction were displayed.

[0049] Figure 3 To verify the association between NPC and oral flora heterotopy in an independent population; a. PCoA diagram based on Bray-Curtis distance reflects the distribution of flora structure in the two parts of NPC patients and the control group (the ellipse is 75% CI); b. The similarity of flora in the paired data of oral and nasopharyngeal flora in NPC patients and the control group (based on Bray-Curtis distance); c. Difference test of nasopharyngeal heterotopy scores of oral flora in NPC patients and the control group (two-group rank sum test).

[0050] Figure 4The AP-PCR agarose gel results of clones from the oral cavity and nasopharynx of the same individual; A. AP-PCR agarose gel results of Fusobacterium nucleatum from two sites of patient S007; B. AP-PCR agarose gel results of Prevotella intermedia from two sites of patients S001, S024 and S027. Clones with the same band pattern are marked with arrows of the same color. F.nuleatum, Fusobacterium nucleatum; P.intermedia, Prevotella intermedia; Fn.a, Fusobacterium nucleatum subsp.Animals; Pi, Prevotella intermedia; Pm, Prevotella melaninogenica; Pm, Prevotella melaninogenica; Ps, Prevotella salivae; Pd, Prevotella denticola; Pn, Prevotellanigrescens; Pj, Prevotella jejuni; Pp, Prevotella pallen.

[0051] Figure 5 Microbiome pathway enrichment analysis revealed significantly enriched pathways between NPC patients and healthy controls.

[0052] Figure 6 The FISH results showed that NPC-enriched bacteria were present in NPC tissues.

[0053] Figure 7 The figure shows the detection of NPC-enriched bacteria in nasopharyngeal tissue RNAseq data.

[0054] Figure 8 The effect of nasopharyngeal carcinoma-enriched bacteria on the tumor microenvironment; A. The distribution of neutrophil enrichment scores in the nasopharyngeal carcinoma-enriched bacteria-positive group (OtoNP+), the nasopharyngeal carcinoma-enriched bacteria-negative group (OtoNP-) and the non-cancer control group, and the t-test was used to analyze the inter-group differences; B. Pathway enrichment analysis showed the enriched signal pathways in the nasopharyngeal carcinoma-enriched bacteria-positive group (OtoNP+) and the nasopharyngeal carcinoma-enriched bacteria-negative group (OtoNP-); B. Based on GO enrichment analysis, C. Based on KEGG enrichment analysis.

[0055] Fig. 9Figure 1. Correlation between oral ectopic bacteria and nasopharyngeal resident bacteria and nasopharyngeal EBV load. (a) Correlation between EBV load and abundance of oral ectopic bacteria in the nasopharynx. According to EBV load, the subjects were divided into 4 groups: undetectable group (EBV was not detected in the sample, N = 78) and low, medium, and high load groups (EBV was detected in the sample, divided according to the 33% and 67% quartiles, N = 25, 25, 25, respectively). Each column represents an individual. EBV load is represented by a dot plot in the upper layer; the corresponding data of the number of oral ectopic bacteria detected are represented by a bar chart in the middle; the bottom heat map shows the relative abundance of oral ectopic bacteria. (b) Relationship between the types of oral ectopic bacteria detected in the nasopharynx and EBV load; P value was determined by Wilcoxon rank sum test. (c) Spearman correlation coefficient of ranked data was used for correlation analysis between oral ectopic bacteria and EBV load, and Wilcoxon rank sum test was used for P value between the two groups. (d) The correlation analysis between the enriched nasopharyngeal resident bacteria and EBV load in the control group was performed using the Spearman correlation coefficient for ranked data, and the P values ​​between the two groups were analyzed using the Wilcoxon rank sum test. *P<0.05, **P<0.01, ***P<0.001.

[0056] Fig.10 In Example 9, Fusobacterium nucleatum promotes the proliferation of nasopharyngeal carcinoma cells.

[0057] Fig.11 In Example 10, Fusobacterium nucleatum promotes the migration ability of nasopharyngeal carcinoma cells.

[0058] Fig.12 In Example 11, Fusobacterium nucleatum promotes the infection of epithelial cells with EB virus.

[0059] Fig.13 In Example 12, Prevotella intermedia promotes the proliferation of nasopharyngeal carcinoma cells.

[0060] Fig.14 In Example 13, Prevotella intermedia promotes the migration ability of nasopharyngeal carcinoma cells.

[0061] Fig.15 In Example 14, Prevotella intermedia can enhance the infection efficiency of epithelial cells to EB virus.

[0062] Fig.16 This is the ROC curve of Prevotella intermedia for the diagnosis of nasopharyngeal carcinoma in Example 15.

[0063] Fig.17 This is the ROC curve of Fusobacterium nucleatum for the diagnosis of nasopharyngeal carcinoma in Example 15.

[0064] Fig.18 This is the ROC curve of the combination of Prevotella intermedia and Fusobacterium nucleatum for the diagnosis of nasopharyngeal carcinoma in Example 15.

[0065] Fig.19 This is the ROC curve of Prevotella intermedia for the diagnosis and validation of nasopharyngeal carcinoma in Example 16.

[0066] Fig. 20 This is the ROC curve of Fusobacterium nucleatum for the diagnosis and validation of nasopharyngeal carcinoma in Example 16.

[0067] Fig.21 This is the ROC curve of the combination of Prevotella intermedia and Fusobacterium nucleatum for the diagnosis and validation of nasopharyngeal carcinoma in Example 16. DETAILED DESCRIPTION

[0068] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0069] Terms and Definitions

[0070] The term "biomarker", also known as "marker" or "diagnostic marker", refers to a measurable indicator of an individual's biological state. Such biomarkers can be any substance in an individual as long as they are related to a specific biological state (e.g., disease state) of the individual being tested, for example, nucleic acid markers (also known as gene markers, such as DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers, species markers (species / genus markers) and functional markers (KO / OG markers), etc. Among them, the meaning of nucleic acid markers is not limited to existing genes that can be expressed as biologically active proteins, but also includes any nucleic acid fragments, which can be DNA or RNA, which can be modified DNA or RNA, or unmodified DNA or RNA, and a collection composed of them. In this article, nucleic acid markers are sometimes also referred to as characteristic fragments. In the present invention, biomarkers can also be represented by "microbial markers" or "bacterial markers". Biomarkers are measured and evaluated, and are often used to examine normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. In a specific embodiment of the present invention, the present invention uses high-throughput sequencing to analyze biological samples of NPC patients and healthy people. Based on the data of high-throughput sequencing, NPC patients and healthy people are compared to determine biomarkers related to the diagnosis and differentiation of NPC patients and healthy people.

[0071] The term "abundance" refers to the measure of the number of target microorganisms in a biological sample. "Abundance" is also referred to as "load". Generally, by molecular methods, the 16S rRNA gene copy number of the target microorganism is typically measured by, for example, fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (qPCR) or PCR / pyrophosphate sequencing, and bacteria are quantified. The quantification of target nucleic acid sequence abundance in a biological sample may be absolute or relative. "Relative quantification" is usually based on one or more internal reference genes, i.e., the 16S rRNA gene from a reference strain, such as using universal primers and expressing the abundance of the target nucleic acid sequence as the percentage of total bacterial 16S rRNA gene copies or by normalization of Escherichia coli 16S rRNA gene copies and measuring bacteria. "Absolute quantification" gives the exact number of target molecules by comparing with a DNA standard or by normalization of DNA concentration. "Quantitative level" can be concentration (DNA amount per unit volume), DNA amount or gene copy number per cell number, cycle threshold value (Ct value) or any mathematical transformation thereof, such as the Log10 of gene copy number.

[0072] In the present invention, any method well known to those skilled in the art can be used to detect microbial markers or determine the level of microbial markers. These methods include, but are not limited to, methods of sequence amplification using primers, and immunological methods using antigen-antibody reactions. Among them, the method of sequence amplification using primers can be, for example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiple PCR, touchdown PCR, hot start PCR, nested PCR, boosted PCR, real-time PCR, differential PCR, cDNA end rapid amplification, reverse polymerase chain reaction, vector-mediated PCR, thermal asymmetric staggered PCR, ligase chain reaction, repair chain reaction, transcription-mediated amplification, autonomous sequence replication, selective amplification reaction of target base sequences. The immunological method using antigen-antibody reactions can be, for example, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, radioimmunodiffusion, Euclid's immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, fluorescence activated cell sorter, protein chip, etc., but the scope of the present invention is not limited thereto.

[0073] The term "sample" refers to a sample containing at least one from a subject. Examples of sample items may be: cells, tissues, body fluids, biopsy specimens, blood, urine, feces, saliva, sputum, plasma, serum, cell culture supernatant, swab samples, etc. In a specific embodiment of the present invention, the sample is a nasopharyngeal swab, nasal lavage fluid, nasopharyngeal lavage fluid, or nasopharyngeal tissue.

[0074] The term "test subject", also referred to as "research subject" or "subject", refers to any animal subject, including but not limited to humans, laboratory animals, livestock and domestic pets. A subject may be inhabited by a variety of microorganisms. A subject may have different microbial communities in various habitats on and in its body. The subject may be diagnosed with a disease or suspected of being at risk for a disease. A subject may have a microbiome state (dysbiosis) that leads to disease. Preferably, the test subject, research subject or subject is a human.

[0075] The term "diagnosis", also known as "early diagnosis", refers to confirming the presence or characteristics of a pathological condition. In a specific embodiment of the present invention, the diagnosis refers to distinguishing the presence or absence of nasopharyngeal carcinoma.

[0076] The term "16S rRNA" refers to rRNA that contains a conserved region common to all species and a hypervariable region that can classify a specific species, which constitutes the 30S subunit of the prokaryotic ribosome. Therefore, the presence of microorganisms can be identified by base sequence analysis. In particular, since there is almost no diversity between homologous species and there is diversity between different species, prokaryotes can be effectively identified by comparing the sequences of 16S rRNA. In addition, since 16S rDNA is a gene that encodes 16S rRNA, 16S rDNA can also be used to identify microorganisms.

[0077] The term "AUC (Area under the ROC curve)" refers to the area under the receiver operating characteristic curve (ROC curve), which is a tool for measuring imbalance in classification. ROC curves and AUC are often used to evaluate the quality of a binary classifier. The ROC curve is a probability curve that plots the relationship between TPR (true positive rate) and FPR (false positive rate) at different thresholds, where the horizontal axis is FPR (False positive rate) and the vertical axis is TPR (True positive rate). FPR refers to how many of all negative examples are predicted as positive examples; TPR refers to how many true positive examples are predicted; the ROC curve can essentially separate "signal" from "noise"; AUC is a probability value. When a positive sample and a negative sample are randomly selected, the probability that the current classification algorithm ranks this positive sample ahead of the negative sample based on the calculated score is the AUC value, which is used as a summary of the ROC curve. The higher the AUC, the better the model's performance in distinguishing positive and negative classes. When AUC = 1, the classifier is able to correctly distinguish all positive and negative points; however, if AUC is 0, the classifier will predict all negatives as positives and all positives as negatives. Therefore, the closer the AUC is to 1, the more sensitive and specific the test is, while an AUC close to 0.5 indicates that the test is neither sensitive nor specific.

[0078] The term "bacterial inhibitor or antibacterial drug" refers to any substance, compound, or combination of substances or compounds that can: (i) inhibit, reduce or prevent the growth of bacteria; (ii) inhibit or reduce the ability of bacteria to establish an infection in a subject; or (iii) inhibit or reduce the ability of bacteria to multiply or remain infectious in the environment. The term "bacterial inhibitor or antibacterial drug" also refers to a compound that can reduce the infectivity or toxicity of bacteria.

[0079] Example 1 Detection of nasopharyngeal flora in nasopharyngeal carcinoma case-control population and discovery of nasopharyngeal carcinoma-related flora characteristics

[0080] This study recruited 70 NPC patients and 86 control subjects, collected nasopharyngeal swab samples and collected clinical and basic information of the participants, extracted DNA from the samples and performed full-length amplicon sequencing of the 16S rRNA gene v1-v9 region (PacBio sequencing technology), and detected the bacterial flora data in the samples. The specific implementation plan is as follows:

[0081] 1.1 Research subjects inclusion and information collection

[0082] This cohort is a case-control study design, which is a group of 70 NPC patients and 86 controls recruited by the applicant's team from June to November 2020 at the Red Cross Hospital of Wuzhou City, Guangxi Zhuang Autonomous Region, China. Pacbio16S full-length sequencing technology was used to explore the characteristics of NPC-related nasopharyngeal microbiota at the species level. The inclusion criteria are as follows:

[0083] *The inclusion criteria for the case group are:

[0084] ① Patients diagnosed with nasopharyngeal carcinoma by pathology and have not received any nasopharyngeal carcinoma-related treatment;

[0085] ②Age 18-75 years old;

[0086] ③ Voluntarily participate, sign the informed consent and accept sample collection.

[0087] *The inclusion criteria for the control group are:

[0088] ①No abnormal symptoms of the head and neck were found;

[0089] ② No serious diseases in the past (such as heart, brain, liver, kidney, mental illness and malignant tumor);

[0090] ③Age 18-75 years old;

[0091] ④ Voluntarily participate, sign the informed consent form and be able to accept sample collection.

[0092] The personal information of the participants was collected by professionally trained staff, including basic personal information such as gender, age, contact information, and personal life history such as medical history, smoking and drinking. The basic information characteristics of the included population are shown in Table 1.

[0093] Table 1: Basic information characteristics of the case-control population

[0094]

[0095] 1.2 Nasopharyngeal swab collection and DNA extraction

[0096] The collection process of nasopharyngeal swab samples is as follows: insert the nasopharyngeal swab into the nasopharynx through the middle nasal meatus, determine the position of the swab with a nasopharyngeal endoscope, evenly wipe the walls and crypts of the nasopharynx for 2-3 circles, take out the swab after it has fully absorbed the wiped tissue, quickly place it in a collection tube filled with preservation fluid, and freeze it in a -80℃ ultra-low temperature refrigerator.

[0097] The DNA extraction process of nasopharyngeal swabs is as follows: thaw the nasopharyngeal swabs on ice, vortex the thawed samples, fully release the exfoliated cells and other substances on the swabs into the preservation solution to make a sample suspension, and then take 200μL of the sample suspension for DNA extraction. Use sterile glass beads to grind at the maximum speed in a vortex instrument for 10 minutes to fully lyse the bacterial cell wall. The pretreated samples are extracted with the QIAGEN tissue and blood DNA extraction kit according to the kit instructions, and the extracted DNA is frozen in a -20℃ refrigerator. Blank control samples are set up during sample processing and DNA extraction.

[0098] 1.3 Sample flora sequencing experimental process

[0099] Nasopharyngeal swab DNA was quantified using a Qubit fluorometer (with a Qubit dsDNA BR kit), and gel electrophoresis was used to control DNA quality. Nasopharyngeal swabs were diluted to 25 ng / μL and dispensed into 96-well plates. Microbiome detection system based on 27F / 1492R primers to amplify the full-length region of the 16S rRNA gene and combined with PacBio third-generation sequencing. The specific process is: the bacterial universal primers 27F (5'-AGR GTT YGA TYM TGG CTC AG-3', Forward primer) and 1492R (5'-RGY TAC CTT GTT ACG ACT T-3', Reverse primer) containing a 12bp barcode sequence were used to amplify the full-length 16S rRNA gene. KAPA HiFi HotStart DNA polymerase was used to amplify the swab DNA for 32-33 cycles.

[0100] *PCR amplification system (total system):

[0101] KAPA HiFi HotStart DNA Polymerase (KAPA)-------12.5μL

[0102] Nuclease-free water(invitrogen)-------4.5μL

[0103] Forward primer(2.5μM)---------------3μL

[0104] Reverse primer(2.5μM)---------------3μL

[0105] Nasopharyngeal swab DNA (2ng / μL)---------------2μL

[0106] *PCR reaction conditions are:

[0107]

[0108] Agarose gel electrophoresis was used to confirm the accuracy of the length of the amplified PCR product (~1500bp); the amplified product was purified using Agencourt AMPure XP (0.6×) magnetic beads, and the purified products of each sample were mixed at equimolar concentrations. The purified products were used to construct the SMRTbell DNA library according to the manufacturer's instructions (PacBio platform) and sequenced using the PacBio Sequel platform (Pacific Biosciences). The sequencing depth of each sample was 5,000 to 20,000 sequences. Negative quality control samples were set up during the PCR and library construction process, and were included in the sequencing together with the blank control samples during the sample processing process.

[0109] 1.4 Analysis process of 16S rRNA full-length amplification sequencing data

[0110] High-quality circular consensus sequences (CCS) were obtained from raw PacBio sequencing data using SMRT Link software (v9.0.0, Pacific Biosciences). Sequences were split into corresponding samples using Lima (v2.0.0) based on the 12 bp barcode sequence on the primers amplified during library construction. The DADA2 (v1.22.0) workflow customized for PacBio full-length 16S rRNA gene sequencing data was used for quality control, denoising, and identification of amplicon sequence variants (ASVs) of CCS sequences. Based on the above process, the number of sequences in each ASV for each sample was obtained. The ASVs obtained from the above process were annotated with bacterial species using the silva_nr99_v138_train_set database combined with the silva_species_assignment_v138 sequence database. According to the “RIDE Checklist”, a series of protocols were applied for sequence quality control and decontamination: specifically, ASVs with annotation information of mitochondria or chloroplasts were removed, and ASVs that were not annotated at the bacterial phylum level were removed. In addition, combined with the sequence information obtained from the negative control samples designed during sample collection and processing and DNA library construction and sequencing, the R package Decontam (v1.10.0) was used to identify and filter potential environmental contamination sequences. The sequencing depth of 2000 was used as the cutoff value, and samples with a sequencing depth lower than this cutoff value were deleted to finally obtain the ASV abundance table after quality control.

[0111] The differences in microbial communities were measured using the Bray-Curtis distance matrix and visualized using principal coordinate analysis (PCoA). Permutational multivariate analysis of variance (PERMANOVA) was used to calculate the difference test in microbial community structure between NPC case and control groups. ANCOM-BC (v1.0.5) was used to analyze the differential species between NPC case and control groups, and the analysis model was corrected for gender, age, smoking, drinking, dental caries, and other oral or nasal diseases. SparCC was used to analyze the co-abundance network of microorganisms, and the results of microbial pairs with significant correlation (correlation coefficient r>0.25 or r<-0.25, and p<0.05) were presented using Cytoscape (v3.9.0).

[0112] 1.5 Results: There are significant differences in the nasopharyngeal flora between NPC patients and healthy controls

[0113] First, we tested whether the nasopharyngeal microbiota structure of NPC patients was significantly different from that of the control population. We performed ANCOM-BC analysis to determine the differentially abundant bacterial species between NPC patients and the control group. As shown in Table 2, at the species level, a total of 20 species with significantly different abundances were identified (FDR-q < 0.05), 15 of which were enriched in the nasopharynx of NPC patients, including potential pathogens such as Prevotella intermedia, Fusobacterium nucleatum, and Peptostreptococcusstomatis; 5 bacteria were significantly reduced, all of which were nasopharyngeal commensal bacteria mainly in the healthy nasopharynx, including Corynebacterium accolens, Staphylococcus epidermidis, and Cutibacterium acnes. At the genus level, 15 genera were found to be enriched in the nasopharynx of NPC patients, and 2 genera were significantly reduced in the nasopharynx of NPC patients, as shown in Table 2 for details.

[0114] Table 2: Species of nasopharyngeal flora with significant differences between NPC cases and controls and their detection rates and abundance information

[0115]

[0116]

[0117] Example 2: Ectopic transmission of oral and nasopharyngeal flora and its risk effects on nasopharyngeal carcinoma

[0118] This example is based on the population described in Example 1, and combined with the paired data of individual nasopharyngeal flora and oral flora from saliva to conduct a joint analysis, and found the association between ectopic transmission of oral and nasopharyngeal flora and nasopharyngeal carcinoma. The specific implementation plan is as follows:

[0119] 2.1 Research subjects inclusion and information collection

[0120] Same as Example 1.

[0121] 2.2 Data Analysis Process

[0122] The bioinformatics processing flow is the same as that in Example 1. For statistical analysis, the rarefaction curve of USEARCH (v10.0.240) was used to evaluate the Alpha diversity analysis. The Bray-Curtis distance matrix was used to measure the differences in microbial communities and visualized by principal coordinate analysis (PCoA). The Bray-Curtis distance information of paired samples from the oral cavity and nasopharynx of the same individual was extracted, and the "1-Bray-Cray-Curtis distance" was used to evaluate the similarity of the flora of the two parts of the same individual. The larger the value, the greater the similarity of the flora of the two parts; the Wilcoxon rank-sum test was used to test the difference in the similarity of the flora between the case group and the control group.

[0123] 2.3 Results: Ectopic transmission of oral and nasopharyngeal flora is a dangerous effect of NPC development

[0124] By analyzing the paired nasopharyngeal and oral microbiota data of 70 NPC patients and 86 controls, we found that the diversity of oral microbiota was significantly higher than that of nasopharyngeal microbiota ( Figure 1 A). Further analysis of the beta diversity of the microbiota revealed that the similarity of the microbiota in the oral cavity and nasopharynx of NPC patients was significantly higher than that in healthy controls (Bray-Curtis distance, Wilcoxon rank sum test, P<0.001, Figure 1 B&1C). We observed that the scores of bacterial ectopic in NPC patients were significantly higher than those in the control group ( Figure 1 D).

[0125] The population was divided into high / low translocation groups with a translocation score of 0.5 as the cutoff value. Compared with individuals with low translocation, individuals with high translocation had a 4.51-fold higher risk of NPC than those with low translocation (95% CI: 1.47-16.04, P = 0.012). Using high and low translocation as grouping variables and the ANCOM-BC algorithm, we found that a total of 33 species were significantly enriched in the high translocation group, including 5 species from Prevotella, 4 species from Streptococcus, and 4 species from Neisseria ( Figure 2 B). In addition, 11 species were significantly reduced in the high-extrabial ectopic group, including 4 species from Corynebacterium, 3 species from Cutibacterium, 3 species from Staphylococcus, and 1 species from Dolosigranulum. The above results indicate that oral microbial ectopic to the nasopharynx is an important risk factor for nasopharyngeal carcinoma.

[0126] Example 3: Verification of the oral to nasopharyngeal flora heterotopia associated with nasopharyngeal carcinoma in an independent population

[0127] In this example, a total of 78 nasopharyngeal carcinoma patients and 38 control subjects were recruited, saliva and nasopharyngeal swab samples were collected, and clinical and basic information of the participants was collected. DNA of the samples was extracted and 16S rRNA gene v4 region amplicon sequencing was performed to detect the flora data in the samples. The specific implementation plan is as follows:

[0128] 3.1 Research subjects inclusion and information collection

[0129] This cohort was designed as a case-control study. 78 NPC patients and 38 controls were recruited from the Cancer Center of Sun Yat-sen University from June 2019 to November 2020. The bacterial flora characteristics in the samples were detected by 16S rRNA gene V4 region amplicon sequencing. The inclusion criteria for the population are as follows:

[0130] *The inclusion criteria for the case group are:

[0131] ① Patients diagnosed with nasopharyngeal carcinoma by pathology and have not received any nasopharyngeal carcinoma-related treatment;

[0132] ②Age 18-75 years old;

[0133] ③ Voluntarily participate, sign the informed consent and accept sample collection.

[0134] *The inclusion criteria for the control group are:

[0135] ①No abnormal symptoms of the head and neck were found;

[0136] ② No serious diseases in the past (such as heart, brain, liver, kidney, mental illness and malignant tumor);

[0137] ③Age 18-75 years old;

[0138] ④ Voluntarily participate, sign the informed consent form and be able to accept sample collection.

[0139] The personal information of the participants was collected by professionally trained staff, including basic personal information such as gender, age, contact information, and personal life history such as medical history, smoking and drinking. The basic information characteristics of the included population are shown in Table 3.

[0140] Table 3: Basic information characteristics of the case-control population

[0141]

[0142]

[0143] 3.2 Sample collection and DNA extraction

[0144] The procedures for saliva and nasopharyngeal swab sample collection and DNA extraction are the same as those in Example 1.

[0145] 3.3 Sample flora sequencing experimental process

[0146] DNA was quantified using a Qubit fluorometer (with the Qubit dsDNA BR kit), and gel electrophoresis was used to control DNA quality. The nasopharyngeal swab was diluted to 25 ng / μL and dispensed into a 96-well plate. Microbiome detection system based on 515F / 806R primer amplification of the 16SrRNA gene V4 region amplicon sequencing. KAPA HiFi HotStart DNA polymerase was used for amplification, 20-22 cycles of amplification of saliva DNA, and 32-33 cycles of amplification of swab DNA. The amplification system and reaction conditions are the same as in Example 1.

[0147] The length of the amplified PCR product was confirmed by agarose gel electrophoresis; the amplified product was purified using Agencourt AMPure XP (0.6×) magnetic beads, and the purified products of each sample were mixed at equimolar concentrations. The purified products were sequenced according to the manufacturer's instructions (Illumina PE250bp platform). The sequencing depth of each sample was 30,000 sequences. Negative quality control samples were set up during the PCR and library construction process, and were included in the sequencing together with the blank control samples during the sample processing process.

[0148] 3.4 Analysis process of 16S rRNA gene bacterial amplicon sequencing data

[0149] For the 16S rRNA V4 region amplicon data, the DADA2 workflow was used for sequence quality control, denoising, and identification of amplicon sequence variants (ASVs). Based on the above process, the sequence quantity data of each sample in each ASV can be obtained. The ASVs obtained by the above process were annotated with bacterial species using the silva_nr99_v138_train_set database. According to the "RIDE Checklist", a series of schemes were applied for sequence quality control and decontamination: specifically, ASVs with annotation information of mitochondria or chloroplasts were removed, and ASVs that were not annotated at the bacterial phylum level were removed. In addition, combined with the sequence information obtained from the negative control samples designed during sample collection and processing and DNA library construction and sequencing, the R package Decontam (v1.10.0) was used to identify and filter potential environmental contamination sequences. The sequencing depth of 5000 was used as the cutoff value, and samples with sequencing depths lower than this cutoff value were deleted, and finally the ASV abundance table after quality control was obtained.

[0150] 3.5 Results: Verification of oral-to-nasopharyngeal bacterial translocation associated with NPC in an independent population

[0151] By analyzing the paired nasopharyngeal and oral flora data of 78 NPC patients and 38 controls, the similarity of the oral and nasopharyngeal flora in NPC patients was significantly higher than that in healthy controls (Bray-Curtis distance, Figure 3 A&3B). The degree of ectopicity was further evaluated by FEAST, and the results showed that the scores of bacterial ectopicity in patients with NPC were significantly higher than those in the control group ( Figure 3 C) The above results indicate that the phenomenon of oral to nasopharyngeal bacterial translocation associated with NPC can also be verified in an independent population.

[0152] Example 4: Culturomics verification of the "oral → nasopharyngeal" ectopic transmission phenomenon of harmful bacteria in NPC

[0153] In order to clarify the ectopic phenomenon of oral and nasopharyngeal flora and common ectopic bacteria, we used the culture group method to isolate the flora from fresh saliva and nasopharyngeal swab samples of 35 independent patients. The specific implementation is as follows:

[0154] 4.1 Population recruitment and sample collection for culture omics

[0155] From April to June 2022, 35 participants were recruited in the nasopharyngeal endoscopy room of the Department of Nasopharyngeal Surgery, Cancer Center of Sun Yat-sen University for this part of the study, including 21 NPC patients and 14 non-NPC controls. For each participant, saliva specimens and nasopharyngeal swab specimens were collected at the same time when they participated. The collection process of nasopharyngeal swabs and saliva is the same as that of Example 1. Sterile reagents and consumables were used during the collection process, and all consumables were irradiated with ultraviolet light for half an hour before use.

[0156] 4.2 Culturomics separation and cultivation

[0157] Fresh saliva and nasopharyngeal swab specimens were immediately placed in an ice box after collection, and sample processing and bacterial culture for culturomics were performed within 1 hour. The swabs were vortexed in an anaerobic PBS solution and prepared into 1:10, 1:100, and 1:100 dilutions. The saliva samples were mixed and diluted to 1:10000, and 50 μL of the dilutions were spread on CBA blood plates with different formulations. For plates with colony formation, 50-100 clones were picked from each sample and spread on new CBA plates according to their aerobic / anaerobic culture conditions. Subseparation of aerobic bacteria was performed in a clean bench, and subseparation of anaerobic bacteria was performed in a glove box. After 2-3 days of subseparation culture, the species of subseparated bacteria were identified by laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For clones whose species information could not be determined by MALDI-TOF-MS, primers 27F (SEQ ID NO: 1, 5'-AGRGTTYGATYMTGGCTCAG-3') and 1492R (SEQ ID NO: 2, 5'-RGYTACCTTGTTACG ACTT-3') were used to amplify the full-length sequence of the 16S rRNA gene and perform Sanger sequencing. The sequences obtained by sequencing were compared with the NCBI database to supplement and verify the results of species identification. After the species information of the clone was clarified, the appropriate culture medium was selected for liquid bacterial growth according to its characteristics. The bacteria were frozen in 25% glycerol during the logarithmic growth period and stored in a -80°C refrigerator for subsequent experiments.

[0158] 4.3 Random Primer PCR

[0159] Random primer PCR (AP-PCR) can form a series of amplified fragments of different lengths by designing random primers and randomly amplifying bacterial genomic DNA through primers. If the genome sequences of two bacteria are the same, the length distribution of the amplified fragments is consistent; conversely, the difference in the length distribution of the amplified fragments of different bacterial clones can be used to determine whether different clones have the same or highly similar genomic sequence information. The AP-PCR primer sequence designed in this experiment is 5'-GAGCGGCCAAAGGGAGCAGAC-3'. The reaction conditions of AP-PCR are:

[0160]

[0161] For the PCR product, 5 μL was taken for agarose gel electrophoresis. The electrophoresis was performed at 150 V for 30 minutes using 1.5% agarose, and then gel imaging was performed.

[0162] 4.4 Whole genome sequencing of a single bacterium

[0163] Single bacterial isolates were cultured in BHI medium at 37°C under anaerobic conditions for 48 hours. DNA was extracted after the bacterial solution became turbid, and the whole bacterial genome was sequenced using Illumina Hiseq 150bp double-end sequencing. The paired data were trimmed using Trimmomatic (v0.39) software, and Bowtie2 (v7.3.0) software was used to align the reference genome of the target bacterial species. The reference genome of Prevotella intermedia is Prevotella intermedia strain ATCC 25611, and the reference genome of Fusobacterium nucleatum is Fusobacterium nucleatum strain ATCC 25586. Based on the alignment file, the average nucleotide identity (ANI) analysis was performed using InStrain (v1.0.0) software. SPAdes (v3.15.5) software was further used to assemble the bacterial whole genome, PROKKA (v1.3) software was used to annotate gene functions, and Roary (v3.11.2) software was used to perform pan-genome analysis to find core genes for phylogenetic analysis.

[0164] 4.5 Results: The harmful oral bacteria found in the above results were only isolated from NPC patients

[0165] First, Columbia blood agar (CBA) plates were used for preliminary experiments of sample culture genomics. The results showed that more than 90% of the bacterial species identified under CBA plate culture were high-abundance bacteria resident in the nasopharynx, such as Propionibacterium, Staphylococcus and Corynebacterium. Since the above-mentioned high-abundance resident bacteria occupy the majority in the nasopharyngeal flora and can survive under aerobic and anaerobic conditions, the isolation and identification of oral ectopic bacteria in the samples was greatly hindered by the clones of a large number of resident bacteria in the culture of unoptimized plates. Therefore, we optimized the culture plates and tested two types of plates, kanamycin-vancomycin CBA and phenethyl CBA. The kanamycin-vancomycin CBA plate can effectively inhibit the growth of nasopharyngeal resident bacteria while not affecting the growth of most ectopic bacteria. Therefore, we finally chose the kanamycin-vancomycin CBA plate for subsequent culture genomics experiments. After cultured according to the process described in 4.2, all participants' nasopharyngeal swab samples were subjected to subsequent species identification if there was clone growth; only the saliva samples of participants with clone growth in the nasopharyngeal swab samples were subjected to species identification, and the saliva samples of participants without clone growth in the nasopharyngeal swab samples were no longer subjected to subsequent species identification. The isolation and identification of bacterial species of the 48 participants are shown in Table 4.

[0166] Among the 34 NPC patients, 5 had cloned growth in their nasopharyngeal swab samples. Fusobacterium nucleatum was isolated from the nasopharyngeal swabs of patients S007 and S038, and the bacteria was also isolated from their oral saliva samples. Prevotella intermedia was isolated from the nasopharyngeal swabs of patients S001, S024, and S027, and Prevotella intermedia and a variety of other Prevotella bacteria were also isolated from their oral saliva samples. There was no cloned growth of the target bacteria in the nasopharynx of the 14 non-NPC control participants.

[0167] Table 4: Basic characteristics of the 48 participants and types of nasopharyngeal / oral isolates cultured by culturomics

[0168]

[0169]

[0170]

[0171] 4.6 Results: The harmful bacteria in the oral cavity and nasopharynx of the same individual are the same strain

[0172] In order to further identify the consistency of the flora in the two parts of the same participant at the strain level, a random primer PCR experiment was designed. The test results showed that for patients S007 and S038 with Fusobacterium nucleatum, two strains with different AP-PCR band distribution were detected in the nasopharynx, and these two strains were also isolated in the saliva samples of the patients; for patients S001, S0024 and S0027 with Prevotella intermedia, one strain of Prevotella intermedia was isolated from their nasopharynx, and Prevotella intermedia was also detected in the oral saliva samples of these patients, and the band distribution was consistent with their own nasopharyngeal strains; in addition, other species of Prevotella were also isolated from the oral cavity of these patients, but they were not isolated in their corresponding nasopharyngeal swab samples. The above research results further demonstrated the phenomenon of flora translocation from the oral cavity to the nasopharynx at the bacterial isolation and strain levels, and this translocation phenomenon showed patient specificity. By calculating the ANI of Prevotella intermedia / Fusobacterium nucleatum isolated from the nasopharynx and oral cavity of the same individual, it was found that the ANI of the isolates from the two sites was greater than 99.999%, while the ANI between isolates from different individuals was approximately 95% to 97% ( Figure 4 C&4D). Through phylogenetic analysis, it was also found that the isolates from two parts of the same individual had a closer genetic evolutionary distance ( Figure 4 E&4F). The above findings further prove the phenomenon of bacterial translocation from oral cavity to nasopharynx at the molecular level.

[0173] Example 5: Enrichment analysis of bacterial flora pathways in nasopharyngeal carcinoma patients

[0174] In order to explore the potential functional association between the discovered nasopharyngeal microbiota markers and nasopharyngeal carcinoma, this example used PICRUSt2 software to predict the community function of the nasopharyngeal microbiota based on the full-length sequence of the 16SrRNA gene, and used LEFSe analysis to find MetaCyc metabolic pathways with significant abundance differences between nasopharyngeal carcinoma patients and controls.

[0175] It was found that pathways related to the synthesis of microbial long-chain fatty acids were significantly enriched in the nasopharyngeal flora of patients with NPC, such as lipid A synthesis pathway, stearic acid synthesis pathway, palmitoleic acid synthesis pathway, etc. ( Figure 5 ). To further explore the potential association between NPC and nasopharyngeal microbiota function, we used the differential species significantly associated with NPC to conduct correlation analysis with metabolic pathways. We found that the bacteria enriched in the nasopharynx of NPC patients were positively correlated with the long-chain fatty acid synthesis pathway, while the bacteria reduced in the nasopharynx were negatively correlated with the long-chain fatty acid synthesis pathway ( Figure 5 The above results suggest that changes in nasopharyngeal flora may affect the occurrence and development of nasopharyngeal carcinoma by affecting the synthesis of long-chain fatty acids in the local microenvironment.

[0176] Example 6: Fluorescence in situ hybridization revealed that nasopharyngeal carcinoma was enriched in tumor tissue

[0177] To further discover the key role of NPC enriched bacterial flora characteristics in the occurrence and development of NPC, this example performed bacterial fluorescence in situ hybridization (FISH) of NPC tumor tissue sections to prove that NPC enriched bacterial flora characteristics can invade and colonize NPC tumors.

[0178] 6.1 Fluorescence in situ hybridization experimental process

[0179] Paraffin-embedded specimens of nasopharyngeal carcinoma tissue were obtained from patients with nasopharyngeal carcinoma, and the slice thickness was 5 μM. The target bacteria for labeling were Fusobacterium nucleatum and Prevotella intermedia. For the labeling of total bacteria, a universal bacterial probe (EUB338, Cy3 labeled) with a sequence of 5′-GCTGCCTCCCGTAGGAGT-3′ was used. For the specific Fusobacterium labeling, a specific probe FITC-5'-atgTTGTCCCTAVCTGTGAGGC-3'-fitc was used; for the Prevotella intermedia labeling, a specific probe FITC-5'-CGTTGCGTGCACTCAAGT C-3'-fitc was used. After defatting and hydration, probe hybridization, washing and other steps, the hybridized and stained tissue slides were observed under an OLYMPUS FV1000 confocal fluorescence microscope to obtain typical images.

[0180] 6.2 Results: NPC-enriched bacteria were detected in tumor tissues

[0181] Bacteria are present in tissue sections of nasopharyngeal carcinoma patients. Fluorescence signals of the above bacteria were observed in sections hybridized with Fusobacterium nucleatum and Prevotella intermedia specific probes; and the fluorescence signals of the target bacteria basically overlap with the universal bacterial marker (EUB338) ( Figure 6 ). This result intuitively demonstrates the presence of the bacterial flora characteristics enriched in nasopharyngeal carcinoma patients found in Examples 2 and 3 in nasopharyngeal carcinoma tumor tissue.

[0182] Example 7: Nasopharyngeal tissue RNAseq sequencing data reveals the detection of NPC-enriched bacteria in NPC tissue and its impact on the tumor microenvironment

[0183] In this example, whole transcriptome sequencing was performed on NPC tumor tissues and NPC non-cancerous tissues to reveal the detection of NPC-enriched bacteria in NPC tissues and their impact on the tumor microenvironment, demonstrating the key role of NPC-related bacterial flora characteristics in the occurrence and development of NPC.

[0184] 7.1 Sample source, sample processing and RNAseq sequencing

[0185] 89 frozen specimens of cancer tissues and 12 nasopharyngeal non-cancerous tissues diagnosed as nasopharyngeal carcinoma by pathology were obtained from the biological sample bank of the Cancer Center of Sun Yat-sen University. RNA was extracted from the above tissue specimens and RNAseq sequencing was performed based on the polyA removal strategy, and the sequencing strategy was Illumina Hiseq 150bp paired-end sequencing.

[0186] 7.2 RNAseq Data Analysis Methods

[0187] In terms of bacterial flora detection, KneadData (v0.10.0) was used to process the raw data and remove low-quality sequences and human sequences. Kraken2 (v2.1.1) was used to identify the species classification level of bacterial flora source data, and Bracken (v2.5.0) was used to calculate the abundance of the identified species. Species with an abundance greater than one in a million were considered detectable. In terms of human sequences, human sequences that passed quality control were aligned with the human reference genome (UCSC hg38) using Hisat2 (v2.1.1), and gene expression information was obtained using htseq-count (v2.0.1). EdgeR (v3.36.0) was used to analyze differentially expressed genes (DEGs), and the adjusted P value <0.05 and |log2FC|>1 were significant cutoff values. ClusterProfilter (v4.2.2) was used to perform gene ontology (GO) biological process (BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on DEGs. Single sample gene set enrichment analysis (ssGSEA) was used to calculate the enrichment scores of immune cell types in tissue microenvironment.

[0188] 7.3 Results: Nasopharyngeal-enriched bacteria can be detected in the cancer tissues of NPC patients

[0189] Figure 7 The detection of 15 NPC-enriched bacteria found in Example 2 in nasopharyngeal tissue is demonstrated, of which 9 NPC-enriched bacteria can be detected in the tissues of NPC patients. Among them, Fusobacterium nucleatum has the highest detection rate in nasopharyngeal tissue, detected in 29.2% of cancerous tissues and 8.3% of non-cancerous tissues. Prevotella intermedia is the second most detected bacterium, detected in 10.1% of cancerous tissues, and no samples were detected in non-cancerous tissues. The above results further verify the presence of NPC-enriched bacteria in NPC tissues.

[0190] 7.4 Results: Intratumoral colonization of NPC-enriched bacteria affects the tumor microenvironment and thus participates in tumorigenesis

[0191] To further evaluate these effects, NPC patients were divided into NPC-enriched bacteria-positive group (OtoNP+, N=29) and NPC-enriched bacteria-negative group (OtoNP-, N=60) according to whether NPC-enriched bacteria were detected in NPC tissues, in order to examine the effect of the presence or absence of NPC-related enriched bacteria in tumor tissues on the immune cell components and intracellular signaling pathways in the tumor.

[0192] ssGSEA analysis found that the neutrophil enrichment score in the positive group tumors was significantly higher than that in the negative group, and the neutrophil enrichment score in tumor tissues was significantly higher than that in non-tumor tissues ( Figure 8 A), indicating that the intratumoral colonization of NPC-enriched bacteria will affect the microenvironment in the tumor, which is mainly composed of neutrophils. In addition, GO enrichment analysis also found that signaling pathways related to myeloid cell chemotaxis, especially neutrophil migration or chemotaxis, were significantly enriched in the positive group, further confirming the association between these bacteria and changes in the tumor microenvironment ( Figure 8 B). Through KEGG signal pathway analysis, a total of 10 significantly different signal pathways were found, among which the interaction pathway between virus-related proteins and cytokines and cytokine receptors was the most significant pathway ( Figure 8 C). The above results indicate that the intratumoral colonization of nasopharyngeal-enriched bacteria affects the tumor microenvironment and thus participates in tumorigenesis.

[0193] Example 8: Oral ectopic bacteria and nasopharyngeal resident bacteria are closely related to nasopharyngeal EB virus load

[0194] To further explore the association between nasopharyngeal EB virus infection and oral ectopic bacteria, we detected the EB virus copy number in nasopharyngeal swab samples of 70 NPC patients and 86 controls in Example 1, and performed an association analysis between the nasopharyngeal EB virus number and oral ectopic bacteria and nasopharyngeal resident bacteria.

[0195] 8.1 EB virus load detection process

[0196] The processing and DNA extraction process of nasopharyngeal swab samples is shown in Example 1. For the extracted nasopharyngeal swab DNA samples, the BamHI-W region of the Epstein-Barr virus genome and the human globin (β-globin) gene are amplified, and β-globin is used as the internal reference gene to calculate the relative load of Epstein-Barr virus in the nasopharyngeal swab. Specifically: Based on the Roche LightCycler 480 fluorescent real-time quantitative PCR system, the probe method is used to amplify the BamHI-W region and β-globin of the Epstein-Barr virus. The applicant's team has established a standard curve based on the BamHI-W region of the Epstein-Barr virus and the β-globin amplification region. After detection, the number of Epstein-Barr virus copies and the number of β-globin gene copies in the sample will be calculated according to the number of amplification cycles (Ct value) and the standard curve on the same reaction plate to obtain the relative load of Epstein-Barr virus in the nasopharyngeal swab (Epstein-Barr virus copies / β-globin copies). The amplification primers and probe sequences used for qRT-PCR are as follows:

[0197]

[0198] 8.2 Statistical Analysis Process

[0199] According to the EB virus load, the absence of an amplification curve in qPCR was defined as no EB virus detected (N = 78); for the 75 samples with an amplification curve, the EB virus copy number values ​​were calculated according to the process described in 8.1, and the samples were divided into a low EB virus load group (N = 25), a medium EB virus load group (N = 25), and a high EB virus load group (N = 25) according to the tertile of the value. For the 13 oral ectopic bacteria found above, the number of species of oral ectopic bacteria detected in each sample was calculated, and the differences between different groups of EB virus load were tested according to the rank sum test (two-sided). For each oral ectopic bacterium, the Spearman correlation method was used to calculate the correlation between its relative abundance and EB virus grouping, and the correlation coefficient R and its corresponding P value were obtained; and the association between the five nasopharyngeal resident bacteria that were significantly reduced in the nasopharynx of NPC patients and the EB virus load was calculated according to this method. To evaluate the association between oral heterotopic bacteria and nasopharyngeal resident bacteria, the median of the relative abundance of each nasopharyngeal resident bacteria was used as the dependent variable to divide the population into low / high corresponding nasopharyngeal resident bacteria groups, and the detection of oral heterotopic bacteria was used as the independent variable to divide the population into with / without corresponding oral heterotopic bacteria groups. A logistic regression model was constructed to evaluate the odds ratio (OR, odds ratio) of oral heterotopic bacteria colonization under different nasopharyngeal resident bacteria abundance groups.

[0200] 8.3 Results: Oral ectopic bacteria were significantly positively correlated with nasopharyngeal EBV load

[0201] The results showed that the nasopharyngeal EB virus load was significantly positively correlated with the types of oral ectopic bacteria that could be detected in the nasopharynx. The types of oral ectopic bacteria in EB virus-positive individuals were significantly higher than those in EB virus-negative individuals, and there was an obvious dose-effect relationship ( Fig. 9 A&9B).

[0202] The abundance of all nasopharyngeal species was correlated with nasopharyngeal EB virus load. A total of 6 species were found to be positively correlated with EB virus, all of which were oral atopic bacteria. Among them, Fusobacterium nucleatum had the strongest correlation with nasopharyngeal EB virus load (R = 0.36, P = 4.2 × 10-6). Other significantly positively correlated species included Peptostreptococcusstomatis, Prevotella intermedia, Gemella morbillorum, Capnocytophaga gingivalis, and Gemella haemolysans ( Fig. 9 C).

[0203] 8.4 Results: There was a significant negative correlation between nasopharyngeal resident bacteria and nasopharyngeal EBV load

[0204] The abundance of all nasopharyngeal species was correlated with the nasopharyngeal EB virus load. A total of four species were found to be negatively correlated with EB virus, and all of them were previously found to be resident bacteria in the nasopharynx that were significantly reduced in NPC patients. They included Corynebacterium accolens (R = -0.39, P = 5.5 × 10 -7 ), Cutibacterium granulosum (R=-0.38, P=1.6×10 -4 ), Cutibacterium acnes (R=-0.32, P=4.2×10 -5 ) and Staphylococcusepidermidis (R=-0.27, P=7.5×10 -4 )( Fig. 9 D).

[0205] Example 9 Fusobacterium nucleatum promotes the proliferation of nasopharyngeal carcinoma cells

[0206] To investigate whether ectopic bacteria Fusobacterium nucleatum affects tumor proliferation, a CCK-8 experiment of the co-culture system of Fusobacterium nucleatum and nasopharyngeal carcinoma cells was performed.

[0207] Specifically: C666 cells (nasopharyngeal carcinoma cell line) were plated in a 96-well plate with a cell density of 4000 cells / well; after the cells were attached to the wall for 12 hours after plating, Fusobacterium nucleatum bacteria were added for co-culture. Fusobacterium nucleatum was cultured to the logarithmic growth phase in an anaerobic environment at 37°C using BHI medium, added to the cells at MOI=100, and cultured in an anaerobic environment for 4 hours. After 4 hours, the co-culture system was washed with PBS, and then complete medium containing 0.06μg / ml metronidazole was added to continue the culture. CCK-8 was used for absorbance detection on the same day, 3 days, and 5 days, and the growth curve was drawn.

[0208] The results showed that compared with the group without Fusobacterium nucleatum treatment, the proliferation ability of cells treated with Fusobacterium nucleatum co-culture was significantly improved ( Fig.10 ). Given that Fusobacterium nucleatum is rarely colonized in the nasopharynx of normal individuals, the presence of Fusobacterium nucleatum may enhance the proliferation ability of nasopharyngeal carcinoma cells.

[0209] To prove the effect of Fusobacterium nucleatum on the proliferation of NPC tumor cells, we heat-inactivated Fusobacterium nucleatum co-cultured with cancer cells and found that the proliferation ability of tumor cells was significantly weakened ( Fig.10 ). The above results indicate that the presence of Fusobacterium nucleatum can enhance the proliferation ability of nasopharyngeal carcinoma cells, and killing Fusobacterium nucleatum will hopefully inhibit the tumor-promoting effect caused by the bacteria.

[0210] Example 10 Fusobacterium nucleatum promotes the migration ability of nasopharyngeal carcinoma cells

[0211] To investigate whether ectopic bacteria Fusobacterium nucleatum affect tumor migration, a transwell experiment of Fusobacterium nucleatum and nasopharyngeal carcinoma cell co-culture system was performed.

[0212] Specifically, HK-1 cells (nasopharyngeal carcinoma cell line) were plated in 6-well plates at a cell density of 4×10 5 cells / well; after 12 hours of plating, Fusobacterium nucleatum bacteria were added for co-culture, and MOI=100, MOI=200 and inactivated groups were set. Culture was carried out in an anaerobic environment for 4 hours. After 4 hours, the co-culture system was washed with PBS, the digested cells were plated in transwell, 60,000 cells were placed in the upper chamber, and metronidazole with a final concentration of 0.06μg / ml was added to the cell culture medium. Crystal violet staining was performed at 37℃5% CO2 for 40 hours to compare the number of perforated cells in each group.

[0213] The results showed that compared with the group without Fusobacterium nucleatum treatment, the migration ability of cells treated with Fusobacteriumnucleatum co-culture was significantly improved ( Fig.11 ).

[0214] To demonstrate the effect of Fusobacterium nucleatum on the migration of NPC tumor cells, we heat-inactivated Fusobacterium nucleatum co-cultured with cancer cells and found that the migration ability of tumor cells was significantly weakened ( Fig.11 ). The above results indicate that the presence of Fusobacterium nucleatum can enhance the migration ability of nasopharyngeal carcinoma cells, and killing Fusobacterium nucleatum will hopefully inhibit the tumor-promoting migration effect caused by the bacteria.

[0215] Example 11 Fusobacterium nucleatum promotes epithelial cell infection with Epstein-Barr virus

[0216] Epstein-Barr virus infection of nasopharyngeal epithelium is an important part of the development of nasopharyngeal carcinoma. To investigate whether ectopic bacteria Fusobacterium nucleatum affect the efficiency of epithelial cell infection with Epstein-Barr virus, free Epstein-Barr virus particles were prepared and Epstein-Barr virus infection experiments were performed in a co-culture system.

[0217] Specifically, 293T cells were plated in a 24-well plate with a cell density of 150,000 cells / well; after 12 hours of plating, Fusobacterium nucleatum bacteria were added for co-culture. Fusobacterium nucleatum was cultured in an anaerobic environment at 37°C using BHI medium to the logarithmic growth phase, added to the cells at MOI = 100, and cultured in an anaerobic environment for 4 hours. After 4 hours, the co-culture system was washed with PBS, and a medium containing antibiotics (double antibiotics and metronidazole) was added. After 24 hours of culture, free EB virus (EB virus: DMEM = 1:1, 150 μl of EBV and 150 μl of DMEM medium were added to the 24-well plate) and incubated for 24 hours. Then, the cells were washed with PBS, trypsinized, and fixed with 4% PFA, and the infection efficiency of EB virus, i.e., the proportion of green fluorescent cells, was detected by flow cytometry.

[0218] The results showed that the proportion of EB virus-positive cells in 293T cells treated with Fusobacterium nucleatum co-culture increased significantly ( Fig.12 ).

[0219] Example 12 Prevotella intermedia promotes the proliferation of nasopharyngeal carcinoma cells

[0220] To investigate whether ectopic bacteria Prevotella intermedia affect tumor proliferation, a CCK-8 experiment of the co-culture system of Prevotella intermedia and nasopharyngeal carcinoma cells was performed.

[0221] Specifically: HK-1 cells (nasopharyngeal carcinoma cell line) were plated in a 96-well plate with a cell density of 1000 cells / well; after the cells were attached to the plate for 12 hours, Prevotella intermedia bacteria were added for co-culture. Prevotella intermedia was cultured in an anaerobic environment at 37°C using BHI medium until the logarithmic growth phase, added to the cells at MOI = 100, and cultured in an anaerobic environment for 2 hours. The co-culture system was then washed with PBS, and complete medium containing 0.6 μg / ml of metronidazole was added for continued culture. CCK-8 was used for absorbance detection on the same day, 2 days, and 3 days, and growth curves were drawn.

[0222] The results showed that compared with the group without Prevotella intermedia treatment, the proliferation ability of cells co-cultured with Prevotella intermedia was significantly improved ( Fig.13 ). Given that the nasopharynx of normal individuals is rarely colonized by Prevotella intermedia, the presence of Prevotella intermedia may enhance the proliferation ability of nasopharyngeal carcinoma cells.

[0223] To prove the effect of Prevotella intermedia on the proliferation of NPC tumor cells, we inactivated the Prevotella intermedia co-cultured with cancer cells by heating and found that the proliferation ability of tumor cells was significantly weakened ( Fig.13 ). The above results indicate that the presence of Prevotella intermedia can enhance the proliferation ability of NPC cells, and killing Prevotella intermedia will hopefully inhibit the effect of promoting tumor proliferation caused by the bacteria.

[0224] Example 13 Prevotella intermedia promotes the migration ability of nasopharyngeal carcinoma cells

[0225] To study whether the ectopic bacteria Prevotella intermedia affect tumor migration, a transwell experiment of the co-culture system of Prevotella intermedia and nasopharyngeal carcinoma cells was performed. Specifically, HK-1 cells (nasopharyngeal carcinoma cell line) were plated in a 6-well plate at a cell density of 4×10 5cells / well; after 12 hours of plating, the cells were attached to the wall, and Prevotella intermedia bacteria were added for co-culture, and MOI = 100 and MOI = 200 groups were set. Culture was carried out in an anaerobic environment for 4 hours. After 4 hours, the co-culture system was washed with PBS, the digested cells were plated in transwell, 60,000 cells were placed in the upper chamber, and metronidazole was added to the cell culture medium at a final concentration of 0.06μg / ml. Crystal violet staining was performed at 37℃5% CO2 for 40 hours to compare the number of perforated cells in each group.

[0226] The results showed that compared with the group without Prevotella intermedia treatment, the migration ability of cells co-cultured with Prevotella intermedia was significantly improved ( Fig.14 ). The above results indicate that the presence of Prevotella intermedia can enhance the migration ability of NPC cells.

[0227] To prove the effect of Prevotella intermedia on the migration of NPC tumor cells, we inactivated the Prevotella intermedia co-cultured with cancer cells by heating and found that the migration ability of tumor cells was significantly weakened ( Fig.14 ). The above results indicate that the presence of Prevotella intermedia can enhance the migration ability of NPC cells, and killing Prevotella intermedia will hopefully inhibit the tumor-promoting migration effect caused by the bacteria.

[0228] Example 14 Prevotella intermedia promotes epithelial cell infection with Epstein-Barr virus

[0229] Epstein-Barr virus infection of nasopharyngeal epithelium is an important part of the development of nasopharyngeal carcinoma. To study whether ectopic bacteria Prevotella intermedia affect the efficiency of epithelial cell infection with Epstein-Barr virus, free Epstein-Barr virus particles were prepared and Epstein-Barr virus infection experiments were performed in a co-culture system.

[0230] Specifically, 293T cells and HK1 cells were plated on a 24-well plate at a cell density of 150,000 cells / well; after the cells were attached to the wall for 12 hours after plating, Prevotella intermedia bacteria were added for co-culture. Prevotella intermedia was cultured to the logarithmic growth phase in an anaerobic environment at 37°C using BHI medium, added to the cells at an MOI of 100, and cultured in an anaerobic environment for 4 hours. After 4 hours, the co-culture system was washed with PBS, and a culture medium containing antibiotics (double antibiotics and metronidazole) was added. After 24 hours of culture, free EB virus (EB virus: DMEM = 1:1, 150 μl of EBV and 150 μl of DMEM culture medium were added to the 24-well plate) and incubated for 24 hours. Subsequently, the cells were washed with PBS, trypsinized, and fixed with 4% PFA. The infection efficiency of EB virus, i.e., the proportion of green fluorescent cells, was detected by flow cytometry.

[0231] The results showed that the fluorescence intensity of 293T cells co-incubated with Prevotella intermedia was significantly higher under EB virus infection than that of the untreated group ( Fig.15 A); ②293T( Fig.15 B) and HK-1( Fig.15 C) After the cell lines were treated with bacteria, the infection rate of EB virus was significantly increased (GFP positive rate detected by flow cytometry). The above results show that Prevotella intermedia can promote the infection of epithelial cells to EB virus.

[0232] Example 15 Diagnostic test for nasopharyngeal carcinoma using Prevotella intermedia and / or Fusobacterium nucleatum as markers

[0233] The population in Example 1 was used for detection and analysis, and whether Prevotella intermedia was detected in the nasopharyngeal swab was used as an evaluation index. The detection method of Prevotella intermedia was as shown in Example 1. Nasopharyngeal carcinoma was diagnosed, and the sensitivity and specificity of diagnosing nasopharyngeal carcinoma were obtained. The results are shown in the following table:

[0234] Grouping Prevotella intermedia negative Prevotella intermedia positive Control group 86 0 Case group 59 11

[0235] It can be calculated that Prevotella intermedia has a high specificity for diagnosing nasopharyngeal carcinoma. None of the 86 control individuals were found to have the bacterium, with a specificity of 100%. The sensitivity of the bacterium is 15.7%, the positive predictive value is 100%, and the negative predictive value is 59.3%. The corresponding ROC curve is shown in Figure 1. Fig.16 shown.

[0236] Using the same samples and methods, and taking the presence of Fusobacterium nucleatum in nasopharyngeal swabs as the evaluation index, the diagnosis results of nasopharyngeal carcinoma are shown in the following table:

[0237] Grouping Fusobacterium nucleatum negative Fusobacterium nucleatum positive Control group 83 3 Case group 53 17

[0238] It can be calculated that Fusobacterium nucleatum has a high specificity for diagnosing nasopharyngeal carcinoma. Only 3 of the 86 control individuals were found to have the bacterium, with a specificity of 96.5%; the sensitivity of the bacterium is 24.3%, the positive predictive value is 85.0%, and the negative predictive value is 61.0%. The ROC curve of Fusobacterium nucleatum is shown in Figure 1. Fig.17 .

[0239] The two bacteria were further used to diagnose nasopharyngeal carcinoma. If any one of the bacteria was detected positive in the nasopharyngeal swab, it was considered positive. The detection results are shown in the following table:

[0240] Grouping Both bacteria were negative Either one of the two bacteria is positive Control group 83 3 Case group 46 24

[0241] The combination of the two bacteria still has a high specificity for diagnosing nasopharyngeal carcinoma, with a specificity of 96.5%; the sensitivity is 34.3%, the positive predictive value is 88.9%, and the negative predictive value is 64.3%. The corresponding ROC curve is as follows Fig.18 shown.

[0242] Example 16 Validation of a diagnostic test for nasopharyngeal carcinoma using Prevotella intermedia and / or Fusobacterium nucleatum as markers

[0243] The diagnostic efficacy of two bacteria, Prevotella intermedia and Fusobacterium nucleatum, in nasopharyngeal carcinoma was further verified in nasopharyngeal tissue.

[0244] The samples and detection methods of this embodiment are the same as those of Example 7. Whether Prevotella intermedia is detected in the nasopharyngeal biopsy tissue is used as an evaluation index. The diagnosis results of nasopharyngeal carcinoma are shown in the following table:

[0245] Grouping Prevotella intermedia negative Prevotella intermedia positive Control group 12 0 Case group 80 9

[0246] It can be calculated that the specificity of Prevotella intermedia for nasopharyngeal carcinoma diagnosis is 100%; the sensitivity of the bacteria is 10.1%. The corresponding ROC curve is as follows Fig.19 shown.

[0247] Taking whether Fusobacterium nucleatum is detected in nasopharyngeal tissue as the evaluation index, the diagnosis results of nasopharyngeal carcinoma are shown in the following table:

[0248] Grouping Fusobacterium nucleatum negative Fusobacterium nucleatum positive Control group 11 1 Case group 63 26

[0249] The results further verified that Fusobacterium nucleatum has a high specificity of 91.7% for diagnosing nasopharyngeal carcinoma; the sensitivity of the bacteria is 29.2%. Fig. 20 shown.

[0250] The two bacteria were further combined, and any one of them was detected positive in the nasopharyngeal tissue, which was considered positive. The detection results are shown in the following table:

[0251] Grouping Both bacteria were negative Either one of the two bacteria is positive Control group 11 1 Case group 62 27

[0252] The results further verified that the combination of the two bacteria still has a high specificity for nasopharyngeal carcinoma diagnosis, with a specificity of 91.7% and a sensitivity of 30.3%. The corresponding ROC curve is shown in Fig.21 shown.

Claims

1. The use of a bacterial detection reagent in the preparation of a product, characterized in that: The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum; The product has one or more of the following functions: A1. Predict the risk of nasopharyngeal carcinoma; A2. Diagnosis or auxiliary diagnosis of nasopharyngeal carcinoma; A3. Predict the risk of recurrence of nasopharyngeal carcinoma; A4. Predict the risk of nasopharyngeal carcinoma metastasis; A5. Predict disease-free survival in patients with nasopharyngeal carcinoma; A6. Predicting distant metastasis-free survival in patients with nasopharyngeal carcinoma; A7. Predict overall survival of patients with nasopharyngeal carcinoma; A8. Prognosis prediction for patients with nasopharyngeal carcinoma.

2. The use according to claim 1, characterized in that The detection reagent is a primer, a probe, an antisense oligonucleotide, an aptamer, and / or an antibody for detecting bacteria; Preferably, the detection reagent is a reagent for detecting bacterial abundance; Preferably, the reagent for detecting bacterial abundance is a 16S rRNA detection reagent, a qPCR detection reagent, an RNAseq detection reagent, a high-throughput sequencing reagent, or a metagenomic sequencing reagent; Preferably, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V1-V9 region; Preferably, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V4 region; Preferably, the reagent for detecting bacterial abundance is a primer; Preferably, the primers are 27F and 1492R primers; Preferably, the sequences of the primers are shown in SEQ ID NOs: 1 and 2.

3. A reagent for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, characterized in that: The detection reagent comprises bacteria, wherein the bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

4. The reagent according to claim 3, characterized in that The reagents are primers, probes, antisense oligonucleotides, aptamers, and / or antibodies for detecting bacteria; Preferably, the detection reagent is a reagent for detecting bacterial abundance; Preferably, the reagent for detecting bacterial abundance is a 16S rRNA detection reagent, a qPCR detection reagent, an RNAseq detection reagent, a high-throughput sequencing reagent, or a metagenomic sequencing reagent; Preferably, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V1-V9 region; Preferably, the reagent for detecting bacterial abundance is a substance for detecting the bacterial 16S rRNA V4 region; Preferably, the reagent for detecting bacterial abundance is a primer; Preferably, the primers are 27F and 1492R primers; Preferably, the sequences of the primers are shown in SEQ ID NOs: 1 and 2; Preferably, the reagents further include extraction reagents of saliva, tissue, nasal mucus, nasal washing fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal washing fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells; Preferably, the reagent is a kit, a test paper and / or a chip.

5. A system for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, characterized in that: include: Information acquisition module: obtaining the bacterial abundance in the subject's sample; Data analysis module: Based on the abundance of bacteria in the subjects' samples, the risk of occurrence, recurrence, and metastasis of nasopharyngeal carcinoma is determined, specifically the probability of developing nasopharyngeal carcinoma, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival; Result output module: output evaluation results; The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

6. The detection system according to claim 5, characterized in that: The information acquisition module comprises the reagent according to any one of claims 3 or 4; Preferably, the judgment criteria of the data analysis module are: if the bacterial abundance is higher than a threshold, the probability of occurrence, recurrence, metastasis risk, poor prognosis, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival of the subject's nasopharyngeal carcinoma is higher than that of a healthy population; If the bacterial abundance is lower than the threshold, the subjects' chances of nasopharyngeal carcinoma development, recurrence, metastasis risk, poor prognosis, shortened disease-free survival, shortened distant metastasis-free survival, and shortened overall survival are lower than those of the general population.

7. An electronic device for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, characterized in that: The electronic device comprises: An acquisition and detection module is configured to acquire a biological sample, detect the biological sample, and obtain the abundance of bacteria in the biological sample; a prevention, diagnosis, and / or prognosis module, wherein the prevention, diagnosis, and / or prognosis module outputs a prevention, diagnosis, and / or prognosis result according to a model based on the bacterial abundance; The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum.

8. The detection system or electronic device according to any one of claims 5 to 7, characterized in that: The sample or biological sample of the subject includes: saliva, tissue, nasal mucus, nasal washing fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal washing fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells; Preferably, the subjects include healthy people and nasopharyngeal carcinoma patients.

9. A method for constructing a model for preventing, diagnosing, and / or prognosing nasopharyngeal carcinoma, characterized in that: The construction method comprises: Testing the biological sample taken from the subject to obtain a bacterial flora dataset; Obtaining the subject's condition of suffering from nasopharyngeal carcinoma, and constructing a first data set using the microbiome data set and the condition of suffering from nasopharyngeal carcinoma; Performing multivariate statistics and data mining on the first data set, performing species difference analysis on the bacterial community data set, and obtaining the bacterial species markers associated with the nasopharyngeal carcinoma; The biological sample includes nasal mucus, nasal washing fluid, nasal swab, nasal exfoliated cells, nasopharyngeal mucus, nasopharyngeal washing fluid, nasopharyngeal swab or nasopharyngeal exfoliated cells; The subjects include healthy people and nasopharyngeal cancer patients; The bacterial strain marker is Prevotella intermedia and / or Fusobacterium nucleatum.

10. Use of a bacterial inhibitor or antibacterial drug in the preparation of a product, characterized in that: The bacteria are Prevotella intermedia and / or Fusobacterium nucleatum; The product has one or more of the following functions: B1. Anti-EB virus; B2. Inhibit the proliferation of nasopharyngeal carcinoma cells; B3. Inhibit the migration of nasopharyngeal carcinoma cells.

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