Cervical and vaginal microbial combinations associated with cervical intraepithelial neoplasia and their applications

Abstract

The present invention discloses a combination of cervical and vaginal microorganisms related to cervical intraepithelial neoplasia and its application. Specifically, the present invention determines the microbial flora with different levels in the cervix of CIN patients by sequencing intestinal microbial samples from individuals with cervical intraepithelial neoplasia (CIN) and healthy individuals, and clarifies the relationship between these microbial flora with different levels and cervical intraepithelial neoplasia (CIN). These cervical microorganisms with different levels can be used to diagnose cervical intraepithelial neoplasia (CIN) diseases.

Description

Technical Field

[0001] The present invention relates to microbiology, and in particular to a combination of cervical and vaginal microorganisms associated with cervical intraepithelial neoplasia and applications thereof. Background Art

[0002] Cervical intraepithelial neoplasia (CIN) is a precancerous lesion characterized by abnormal cell growth on the squamous-columnar junction surface of the cervix. Depending on the extent of the affected tissue, CIN can be divided into CIN 1 (low-grade neoplasia), CIN 2 and CIN 3 (the most severe form). Although low-grade dysplasia (CIN 1) can subside, it can progress to high-grade dysplasia (CIN3), and even more seriously progress to cervical cancer. Known CIN risk factors include premature sexual life, multiple sexual partners, smoking, infection of sexually transmitted viruses, and a history of vulvar and anal dysplasia. Persistent infection with human papillomavirus (HPV), especially persistent infection with high-risk HPV-16 and HPV-18 subtypes, is the main risk factor involved in the development of CIN. HPV can induce invasion through viral proteins E6 and E7, change cell proliferation and differentiation and promote malignant transformation. On the other hand, HPV can induce local damage and damage the normal antibacterial microenvironment of the reproductive tract by damaging the innate and adaptive immunity of the cervical mucosa.

[0003] Commensal microbial communities contribute to the maintenance of homeostasis, regulating the host immune system and metabolism. Disturbances in microbial communities, known as dysbiosis, contribute to many complex human diseases, such as metabolic diseases, gastrointestinal diseases, and cancer. Considered as environmental factors, microbial communities can produce more than four million products to induce metabolic infections in the low-grade inflammation that characterizes metabolic diseases. For example, increased abundance of Atopobium mvaginae and Porphyromonas spp. was found in women with cancer. However, research on the relationship between vaginal and cervical microbiota and its role in CIN risk is limited.

[0004] There is currently no very effective early detection method for cervical intraepithelial neoplasia (CIN) in clinical practice. Therefore, it is urgent to establish a highly sensitive and economical method to meet the needs of clinical diagnosis, prevention or treatment of cervical intraepithelial neoplasia (CIN). Summary of the invention

[0005] The purpose of the present invention is to provide a cervical and vaginal microbiome associated with cervical intraepithelial neoplasia, which can serve as an early marker of cervical intraepithelial neoplasia.

[0006] In a first aspect of the present invention, a microbial combination is provided, comprising one or more microorganisms selected from the group consisting of Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0007] In another preferred embodiment, the microbial combination at least includes: Peredibacter and Ralstonia.

[0008] In another preferred embodiment, the microbial combination further comprises one or more microorganisms selected from the group consisting of Brevundimonas and enhydrobacter.

[0009] In another preferred embodiment, the microbial combination further includes: Pseudomonas.

[0010] In another preferred embodiment, the microbial combination further comprises: Brevundimonas and enhydrobacter.

[0011] In another preferred embodiment, the microbial combination is derived from a cervical sample (such as a cervical swab).

[0012] In another preferred embodiment, the microbial combination is used for the diagnosis (including early diagnosis and / or auxiliary diagnosis) and treatment (including auxiliary treatment) of cervical intraepithelial neoplasia (CIN); or is used to prepare a kit or reagent, wherein the kit or reagent is used for the diagnosis (including early diagnosis and / or auxiliary diagnosis) and treatment (including auxiliary treatment) of cervical intraepithelial neoplasia (CIN).

[0013] In another preferred embodiment, each microbial biomarker is detected by PCR; preferably, the PCR includes QPCR or RT-QPCR.

[0014] In another preferred embodiment, the microbial combination is a gene combination, and the gene combination comprises specific gene nucleic acid fragments (such as 16S rRNA gene sequences) of each microorganism.

[0015] The second aspect of the present invention provides a reagent set for diagnosing (including early diagnosis and / or auxiliary diagnosis) cervical intraepithelial neoplasia (CIN), wherein the reagent set includes reagents for detecting each microorganism in the microbial combination as described in the first aspect of the present invention.

[0016] In another preferred embodiment, each of the reagents is a primer, probe, antisense oligonucleotide, aptamer or antibody specific to each microorganism.

[0017] In another preferred embodiment, the reagent is a PCR detection reagent; preferably, it includes primers that specifically amplify each microbial gene; more preferably, it also includes a probe that specifically binds to each microbial gene.

[0018] The third aspect of the present invention provides a kit, wherein the kit comprises the microbial combination as described in the first aspect of the present invention, and / or the reagent set as described in the second aspect of the present invention.

[0019] In another preferred embodiment, each microorganism in the microbial combination as described in the first aspect of the present invention is used as a standard.

[0020] In another preferred embodiment, the kit further comprises a PCR buffer and / or a PCR reaction enzyme system.

[0021] In another preferred embodiment, the microbial combination is a gene combination, and the gene combination contains specific gene nucleic acid fragments of each microorganism.

[0022] In a fourth aspect of the present invention, there is provided a use of a microbial combination or a detection reagent thereof for preparing a kit for diagnosing (including early diagnosis and / or auxiliary diagnosis) cervical intraepithelial neoplasia (CIN), wherein the microbial combination comprises one or more microorganisms selected from the group consisting of Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0023] In another preferred embodiment, the microbial combination at least includes: Peredibacter and Ralstonia.

[0024] In another preferred embodiment, the microbial combination further comprises one or more microorganisms selected from the group consisting of Brevundimonas and enhydrobacter.

[0025] In another preferred embodiment, the microbial combination further includes: Pseudomonas.

[0026] In another preferred embodiment, the microbial combination further comprises: Brevundimonas and enhydrobacter.

[0027] In another preferred embodiment, the diagnosis comprises the steps of:

[0028] (1) providing a sample from the cervix of a subject to be tested, and detecting the level of each microorganism in the microbial combination in the sample by a PCR method;

[0029] (2) Compare the level measured in step (1) with the corresponding microbial level in the normal population.

[0030] In another preferred embodiment, in step (1), the 16S rDNA of each microorganism is detected using the PCR method.

[0031] In a fifth aspect, the present invention provides a drug for treating (including adjuvant treatment) cervical intraepithelial neoplasia (CIN), wherein the drug has the ability to regulate the microbial combination described in the first aspect of the present invention in the patient's cervix.

[0032] In a sixth aspect of the present invention, there is provided a method for screening a drug for treating (including adjuvant treatment of) cervical intraepithelial neoplasia (CIN), the method comprising the steps of:

[0033] (1) In a test group, a test compound is administered to a subject to be tested, and the level L1 of each microorganism in the microbial combination in a sample derived from the subject in the test group is detected; in a control group, a blank control (including a solvent) is administered to a subject to be tested, and the level L2 of each microorganism in the microbial combination in a sample derived from the subject in the control group is detected;

[0034] (2) comparing the level L1 and the level L2 detected in the previous step to determine whether the test compound is a candidate compound for treating cervical intraepithelial neoplasia (CIN);

[0035] Wherein, the microbial combination includes one or more microorganisms selected from the following group: Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0036] In another preferred embodiment, the microbial combination at least includes: Peredibacter and Ralstonia.

[0037] In another preferred embodiment, the microbial combination further comprises one or more microorganisms selected from the group consisting of Brevundimonas and enhydrobacter.

[0038] In another preferred embodiment, the microbial combination further includes: Pseudomonas.

[0039] In another preferred embodiment, the microbial combination further comprises: Brevundimonas and enhydrobacter.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A-1F Changes in vaginal and cervical microbial diversity associated with CIN are shown:

[0042] Figure 1A : Study design;

[0043] Figure 1B : Microbial diversity associated with CIN in the vagina and cervix;

[0044] Figure 1C : Comparison of vaginal and cervical microbial diversity;

[0045] Figure 1D :Microbial diversity between vagina and cervix in different CIN subtypes;

[0046] Figure 1E : PCoA results of microbial communities;

[0047] Figure 1F : Correlation between PCoA1 and the onset of cervical sexual activity. DETAILED DESCRIPTION

[0048] The inventors of the present invention have conducted in-depth research and determined the differences in the levels of various cervical microorganisms present in patients with cervical intraepithelial neoplasia (CIN) by sequencing 16s rRNA of cervical and vaginal microbial samples from individuals with cervical intraepithelial neoplasia (CIN) and healthy individuals, and determined the relationship between these level differences and cervical intraepithelial neoplasia (CIN). These intestinal microorganisms with level differences can be used in compositions or kits for preventing, diagnosing / treating cervical intraepithelial neoplasia (CIN) diseases.

[0049] The present invention confirms that the classification of cervical microbial populations in CIN patients is significantly different from that in healthy people, and there are differences at the levels of multiple microorganisms (genus) (P<0.05). The abundance of the following microbial genera in patient samples is more than twice that of healthy people, and the difference is statistically significant, suggesting that the levels of these microbial genera are closely related to the onset of cervical intraepithelial neoplasia (CIN): Pseudomonas, Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0050] The present invention provides the use of a microbial combination for preparing or screening a cervical intraepithelial neoplasia (CIN) detection product, wherein the microbial combination comprises one or more microorganisms selected from the following group: Pseudomonas, Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0051] Ralstonia, Peredibacter, Brevundimonas, and enhydrobacter.

[0052] In another preferred embodiment, the microbial combination at least includes: Peredibacter and Ralstonia.

[0053] In another preferred embodiment, the microbial combination further comprises one or more microorganisms selected from the group consisting of Brevundimonas and enhydrobacter.

[0054] In another preferred embodiment, the microbial combination further includes: Pseudomonas.

[0055] In another preferred embodiment, the microbial combination further comprises: Brevundimonas and enhydrobacter.

[0056] In another preferred embodiment, the microbial combination includes at least: Pseudomonas and Ralstonia.

[0057] In another preferred embodiment, the microbial combination further comprises one or more microorganisms selected from the group consisting of Peredibacter, Brevundimonas, and enhydrobacter.

[0058] In another preferred embodiment, the microbial combination further includes: Peredibacter.

[0059] In another preferred embodiment, the microbial combination further comprises: Brevundimonas and enhydrobacter.

[0060] The present inventors have found that in patients with cervical intraepithelial neoplasia (CIN), the abundance levels of certain microorganisms in the cervix of the patients are significantly increased, while the abundance levels of certain microorganisms in the cervix of the patients are significantly decreased.

[0061] For example, the level of Peredibacter in the cervix of patients with cervical intraepithelial neoplasia (CIN) is more than 4 times the level of Peredibacter in healthy people;

[0062] The level of Pseudomonas in the cervix of patients with cervical intraepithelial neoplasia (CIN) is more than 4 times higher than that of healthy people;

[0063] The level of Brevundimonas in the cervix of patients with cervical intraepithelial neoplasia (CIN) is more than twice that of Brevundimonas in healthy people.

[0064] For example, the level of Ralstonia in the cervix of patients with cervical intraepithelial neoplasia (CIN) is less than 1 / 4 of the level of Ralstonia in healthy people;

[0065] The level of enhydrobacter in the cervix of patients with cervical intraepithelial neoplasia (CIN) is less than 1 / 2 of the level in healthy people.

[0066] Therefore, the intestinal microorganisms can be used as biomarkers for the diagnosis and detection of cervical intraepithelial neoplasia (CIN).

[0067] In addition, the present invention also found that the levels of the following microorganisms in the cervix of patients with cervical intraepithelial neoplasia (CIN) were significantly different from those in healthy people (increased by more than 1 times):

[0068] Rhizobium, Curvibacter, Aquabacterium, Granulicatella.

[0069] In one embodiment, the cervical intraepithelial neoplasia (CIN) detection product includes a microbial abundance detection reagent for the microorganism; preferably a specific identification reagent for each microorganism.

[0070] In one embodiment, the specific recognition reagent is selected from a specific primer, a probe, an antisense oligonucleotide, an aptamer or an antibody for each of the microorganisms.

[0071] In one embodiment, the specific primers are capable of detecting 16S rRNA of each microorganism and other primers that can identify gene sequences of the specific microorganism.

[0072] "16S rRNA" refers to the rRNA that constitutes the 30S small subunit of the prokaryotic ribosome. On the one hand, most of its base sequence is highly conserved, and on the other hand, some regions show high base sequence diversity. In particular, there is almost no diversity between the same species, but there is diversity between different species. Therefore, by comparing the 16S rRNA sequence, prokaryotes can be effectively identified. The 16S rRNA gene sequence of a specific microorganism can be retrieved and obtained from a public database.

[0073] The 16s rRNA gene sequences of representative microorganisms of the present invention are as follows:

[0074] >Pseudomonas (SEQ ID NO.1)

[0075]

[0076] >Brevundimonas(SEQ ID NO.2)

[0077]

[0078] >Ralstonia(SEQ ID NO.3)

[0079]

[0080] >Peredibacter(SEQ ID NO.4)

[0081]

[0082] >Enhydrobacter(SEQ ID NO.5)

[0083]

[0084] In the present invention, the term "primer" is a sequence of 7 to 50 bp that can form a base pair complementary to a template chain and acts as a starting point for replicating the template chain. Primers are usually synthesized, but naturally occurring nucleic acids can also be used. The sequence of the primer does not necessarily need to be exactly the same as the sequence of the template, as long as it is sufficiently complementary and can hybridize with the template. Additional features that do not change the basic properties of the primer can be mixed in. Examples of additional features that can be mixed in include methylation, capping, replacement of more than one nucleic acid by a homolog, and modification between nucleic acids, but are not limited to these.

[0085] As an embodiment, in the present invention, the above primers can be used to amplify the 16SrRNA of the corresponding microorganism and other gene sequences that can identify the microorganism. After amplifying the sequence, the presence of the microorganism can be detected or the level of the microorganism can be measured by whether the desired product is generated.

[0086] The sequence amplification method using primers can use various methods known in the art. For example, polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, nested PCR, real-time fluorescence quantitative PCR, etc. can be used.

[0087] The specific identification reagent combination of intestinal microorganisms provided by the present invention can be used to prepare a cervical intraepithelial neoplasia (CIN) detection kit. Preferably, the kit is a PCR detection kit; the kit may include a test tube or other suitable container, a reaction buffer, triphosphate deoxynucleotides (dNTPs), Taq-polymerase, SYBR Green fluorescent dye, DEPC-water, etc.

[0088] The present invention also provides a gene chip for detecting cervical intraepithelial neoplasia (CIN), wherein the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe specifically recognizes the gene sequence of each microorganism in the microorganism combination.

[0089] In the present invention, the term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

[0090] Under normal circumstances, those skilled in the art can design primers and probe sequences that can specifically detect the target sequence based on known target sequence information.

[0091] The probe has a base sequence complementary to a specific base sequence of the target gene. Here, "complementary" means hybridization, and may not be completely complementary. These polynucleotides usually have a homology of 90% or more, preferably 95% or more, and more preferably 100% with respect to the specific base sequence. These probes may be DNA or RNA, or may be polynucleotides in which nucleotides in part or all of them are replaced by artificial nucleic acids such as PNA (Polyamide nucleic acid), LNA (locked nucleic acid, Bridged Nucleic Acid), ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids), GNA (Glycerol nucleic acid), TNA (Threose nucleic acid), etc.

[0092] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as the conditions described in "Molecular Cloning Laboratory Guide" by Sambrook.J et al. (Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0093] Example 1 Study subjects and sample collection

[0094] The study was approved by the hospital's ethics committee and was conducted in accordance with the principles described in the Declaration of Helsinki. A total of 60 women diagnosed with cervical intraepithelial neoplasia (CIN) by histopathology were recruited in this study. Sixty age- and region-matched healthy participants were recruited from the hospital's physical examination center as a control group. All healthy participants were excluded from CIN by Pap smear and colposcopy. Clinical characteristics such as BMI, sexual activity, and sexual partners were collected. Written informed consent was obtained from each participant.

[0095] Samples were collected from the cervix and mid-vagina of each participant. For each participant who underwent vaginal endoscopy, the cotton swab was gently rotated for 10 seconds to fully collect microbial samples located in the mid-to-upper vagina and cervix. To avoid contamination by microorganisms from the lower vagina, the cotton swab was placed in a tube to isolate it from the human body. The samples were placed in a sterile container and kept at 4°C for up to 4 hours before processing, or immediately frozen and stored at -80°C until further use.

[0096] Example 2 16S rRNA analysis of sample microbial communities

[0097] DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. DNA quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and a qubit fluorometer (Life, Waltham, MA).

[0098] 16S rRNA gene sequencing

[0099] For bacterial profiling, polymerase chain reaction (PCR) amplification was performed using region-specific primers (338F-806R) targeting the V3-V4 variable region of the 16S rRNA gene.

[0100] The primer pairs are:

[0101] 5'-ACTCCTACGGGAGGCAGCA-3' (forward, SEQ ID NO. 6) and;

[0102] 5'-GGACTACHVGGGTWTCTAAT-3' (reverse, SEQ ID NO. 7).

[0103] Libraries were prepared using the TruSeq Nano DNA LT Library Prep Kit (FC-121-4001, Illumina, San Diego, CA) (FC-121-4001, Illumina, San Diego, CA) and purified using AMPure XP purification beads (Beckman Coulter, Brea, CA). Amplicon libraries were quality assessed by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) and quantified by Promega QuantiFluor. Libraries with unimodal peak distribution and sufficient concentration (>2 nM) were pooled and sequenced on the MiSeq platform (Illumina, San Diego, CA) to generate 2×300 bp paired-end reads.

[0104] 16S rRNA amplicon sequence analysis

[0105] The raw sequencing data were filtered based on sequencing quality using Trimmomatic software. Clean reads were merged into fragments according to the overlap between the reads and their counterparts. Then, the merged sequences were clustered into operational taxonomic units (OTUs) with a 97% identity threshold using the Unoise algorithm by using the VSEARCH program. The taxonomic classification of OTU was performed based on the EzBioCloud reference database (version 2017.10) using the UCLUST algorithm in QIIME (V1.9). Low-abundance OTUs (<0.01% relative abundance) were filtered out. The sequencing depth was adjusted proportionally to a fixed value of 20,000 sequences using the VSEARCH program, and the OTU table was standardized proportionally. A phylogenetic tree for standardizing OTUs was constructed using the FastTree algorithm implemented in the QIIME software. Sparse α diversity was assessed by rarefaction curves, which were from 500 to 20,000 sequence depths. Alpha diversity was calculated using the standardized OTU table. Paired Bray-Curtis distances were calculated to study the similarity of microbial communities between different groups. The summarize_taxa.py script in QIIME was used to summarize the relative abundance of different taxonomic levels (phylum, class, order, family, and genus) based on the standardized OTU table.

[0106] Statistical analysis

[0107] Statistical analyses and data visualization were performed in R (v3.6). Beta diversity was assessed using principal coordinating analysis (PCoA) and permutation multivariate analysis of variance (PERMANOVA). Relative abundances were arcsine square transformed for visualization. Wilcoxon rank-sum test was used to detect differentially abundant OTUs and taxa. Multivariate analysis of linear models (MaAsLin) was also applied to correct for covariates and identify differentially abundant taxa. The Q-value package was used in the MaAsLin analysis. P values ​​were corrected for multiple testing using the false discovery rate (FDR). Correlation analysis was performed using the Spearman coefficient. An FDR of 0.05 was used as a significance cutoff.

[0108] result

[0109] Object characteristics

[0110] A total of 60 women diagnosed with CIN (denoted as CIN) and 60 healthy women (denoted as HC) matched for age and region were included in this study. The median age of the CIN group and the control group was 40.00 years and 38.00 years, respectively. No significant differences were found for BMI (P = 0.198), time of onset of sexual activity (P = 0.064), and proportion of multiple sexual partners (P = 0.741). A significantly higher HPV positivity rate was observed in the CIN group compared with the control group (89.5% vs 57.9%, P < 0.001). Among the CIN patients, 68.3% of the patients were classified as CIN 1, 10% were classified as CIN 2, and 21.7% were classified as CIN 3.

[0111] Dysbiosis of the cervical microbiota associated with CIN

[0112] For each participant, cervical and mid-upper vaginal samples were collected from CIN patients (CIN) and healthy controls (HC) for pairwise comparison of microbiome changes in the women’s reproductive tract. Figure 1A After quality control, 4 HC samples and 15 CIN samples were filtered. We first observed changes in microbial diversity across the vagina and cervix between CIN and HC ( Figure 1B Unfortunately, although slightly greater OTU counts were observed in the CIN group than in the HC group in cervical samples (mean counts: 71.9 vs 58.3, P = 0.031), no significant changes in Shannon diversity were found in the CIN group (P = 0.172 in the vagina and P = 0.226 in the cervix, respectively, Figure 1B However, in the CIN group, the microbial diversity of cervical samples was increased relative to their paired vaginal samples (P = 1.4 × 10 -4 , Figure 1C In addition, subtype analysis of the CIN group showed that the Shannon diversity of the cervical microbiome was significantly increased in the CIN 1 subtype (P = 3.98 × 10 -4 , Figure 1D ).

[0113] To characterize the vaginal and cervical microbial communities in CIN, we then calculated the Bray-Curtis (BC) distance between each sample based on OTU level abundance ( Figure 1E ). Under the sample size setting of the present invention, no significant community differences were found between HC and CIN across the vagina or cervix (AdnossP=0.368, Figure 1EFurther exploration of the relationship between the first component of the main ordinal analysis (PCoA1) and clinical variables identified a significant negative correlation between PCoA1 and the onset of sexual activity in the cervical microbiota in CIN (Spearman's r = -0.39, P = 0.0042, Figure 1F In conclusion, our results demonstrate increased changes in microbial diversity in the cervix of patients with CIN and suggest that clinical risk factors for CIN may modulate the cervical microbiome.

[0114] Identification of characteristic microorganisms involved in CIN groups

[0115] To further characterize the perturbed microbiota associated with CIN, the differential abundance of OTUs and taxonomic units present at the continuous level (e.g., phylum, family, and genus) was examined. The results showed that for women with CIN, the differential abundance of OTUs in the cervical microbiome was significantly different from that in the vaginal microbiome.

[0116] Although the reduced abundance of Actinomycetes-related OTUs in vaginal samples was significantly associated with CIN, the enrichment of Proteobacteria-related OTUs in cervical samples characterized CIN-related dysbiosis. Although Lactobacillus-related OTUs were reduced, Gardnerella-related OTUs were also significantly reduced in the vagina (log2FC = -0.60, P = 0.0009).

[0117] By comparing the top OTUs in the vagina and cervix, we found that the direction of change between the two locations was very consistent (Spearman r = 0.7, P = 1.20 × 10- 6 However, only 3 OTUs (2 from Stenotrophomonas and 1 from Porphyrobacter) were consistently significant in both the vagina and cervix (P < 0.01), indicating independent signals for the respective locations.

[0118] Next, representative microbial taxa of the altered vaginal and cervical microbiota associated with CIN were identified, and OTUs belonging to the same taxonomic units were clustered.

[0119] Similar to the results at the OTU level, we observed only a limited number of differentially abundant species in the vagina. Finegoldia, Prevotella, and KQ9578_g (a species of Luminococcus) were negatively associated with CIN, while Porphyrobacter, Blautaia, and Rhodococcus were upregulated in CIN.

[0120] However, bacterial communities significantly associated with CIN were identified in the cervical microbiota (Table 2). Multivariate analysis was further performed using a linear model (MaAsLin) to adjust for potential covariates such as age, BMI, and HPV infection. The linear model identified dominant bacteria significantly associated with CIN in the cervix (Table 2).

[0121] Table 1. Baseline characteristics of the study subjects

[0122]

[0123] Abbreviations: Q1: first quartile; Q3: third quartile; SD: standard deviation; CIN: cervical intraepithelial neoplasia.

[0124] Table 2. Differences in abundance of microbial genera associated with CIN in the cervix detected by multivariate analysis

[0125]

[0126] a Average relative abundance of genera in HC and CIN expressed as percentage (%).

[0127] Abbreviations: P: Univariate P value obtained from the Wilocxon rank-sum test. P m : Multivariate P values ​​were derived from MaAsLin, adjusted for age, BMI, time since sexual activity onset, and HPV infection. mad : FDR-corrected multivariate P-values ​​to account for multiple testing.

[0128] Example 3 Clinical Detection and Verification of Cervical Microflora in CIN Patients

[0129] The above studies show that the species classification of cervical flora in CIN patients is significantly different from that in healthy people, and there are differences in the levels of multiple microorganisms. The levels of Peredibacter, Pseudomonas, and Brevundimonas in patients are significantly higher than those in healthy people (P<0.05); the levels of Ralstonia and enhydrobacter in patients are significantly lower than those in healthy people (P<0.05).

[0130] According to the correlation between CIN and related flora, CIN can be diagnosed by detecting the abundance of related flora in cervical samples. In this example, 100 cervical samples from healthy patients and CIN patients were collected, and the samples were numbered and randomly tested. Specific fluorescent quantitative PCR primer probes were designed for the 16S rRNA gene sequence of the target microorganism, and an internal standard was set for microbial level detection.

[0131] Fluorescence quantitative PCR amplification detection system: 0.5 μl of upstream and downstream primers of each strain, 5 μl of template DNA, 6 μl of SYBR Green. Fluorescence quantitative PCR reaction conditions are: 95°C for 10 min, 95°C for 15 s, 60°C for 2 min, for a total of 40 cycles.

[0132] An internal standard gene and a pure water control sample were set for each sample.

[0133] The PCR reaction was carried out on an ABI7500 fluorescent real-time quantitative PCR instrument, and the ΔΔCT method (CT value comparison method) was used to calculate the expression abundance of each target gene in the sample.

[0134] The results of testing 100 cervical samples from the above-mentioned healthy individuals and CIN patients showed that the levels of the above-mentioned microbial flora in the intestinal flora of CIN patients were significantly different from those of healthy individuals (P<0.05). This result was consistent with the results of 16SrRNA library sequencing. The results showed that the detected flora had high sensitivity and specificity, and therefore could be used for auxiliary diagnosis of CIN patients.

[0135] The logistic regression model was used for analysis, and the ROC curve was drawn to consider the diagnostic performance of the strains. The ROC curve showed that the markers of Peredibacter, Brevundimonas, enhydrobacter, Pseudomonas, and Ralstonia all had great diagnostic value and could meet the needs of clinical auxiliary diagnosis.

[0136] Therefore, when one or more indicators of the above-mentioned microbial flora of the cervical flora of the tested population are different from those of normal people, the probability of suffering from cervical intraepithelial neoplasia (CIN) is higher, which helps to assist in the diagnosis of the disease condition and is also a potential target for the treatment of cervical intraepithelial neoplasia (CIN).

[0137] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Zhangpu County Hospital <120> Cervical and vaginal microbial combinations associated with cervical intraepithelial neoplasia and their applications <130> P210609 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 1458 <212> DNA <213> Pseudomonas <400> 1 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgaaggg agcttgctcc 60 tggattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggataac 120 gtccggaaac gggcgctaat accgcatacg tcctgaggga gaaagtgggg gatcttcgga 180 cctcacgcta tcagatgagc ctaggtcgga ttagctagtt ggtggggtaa aggcctacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcagtaagt taataccttg ctgttttgac gttaccaaca gaataagcac cggctaactt 480 cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta atcggaatta ctgggcgtaa 540 agcgcgcgta ggtggttcag caagttggat gtgaaatccc cgggctcaac ctgggaactg 600 catccaaaac tactgagcta gagtacggta gagggtggtg gaatttcctg tgtagcggtg 660 aaatgcgtag atataggaag gaacaccagt ggcgaaggcg accacctgga ctgatactga 720 cactgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780 aaacgatgtc gactagccgt tgggatcctt gagatcttag tggcgcagct aacgcgataa 840 gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 900 cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta cctggccttg 960 acatgctgag aactttccag agatggattg gtgccttcgg gaactcagac acaggtgctg 1020 catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgtaac gagcgcaacc 1080 cttgtcctta gttaccagca cctcgggtgg gcactctaag gagactgccg gtgacaaacc 1140 ggaggaaggt ggggatgacg tcaagtcatc atggccctta cggccagggc tacacacgtg 1200 ctacaatggt cggtacaaag ggttgccaag ccgcgaggtg gagctaatcc cataaaaccg 1260 atcgtagtcc ggatcgcagt ctgcaactcg actgcgtgaa gtcggaatcg ctagtaatcg 1320 tgaatcagaa tgtcacggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca 1380 tgggagtggg ttgctccaga agtagctagt ctaaccgcaa gggggacggt taccacggag 1440 tgattcatga ctggggtg 1458 <210> 2 <211> 1385 <212> DNA <213> Brevundimonas <400> 2 agcgaacgct ggcggcaggc ctaacacatg caagtcgaac ggactcttcg gagttagtgg 60 cggacgggtg agtaacacgt gggaacgtgc ctttaggttc ggaataagcc cgggaaactg 120 ggtctaatac cgaatgtgcc cttcggggga aagatttatc gcctttagag cggcccgcgt 180 ctgattagct agttggtagt gtaatggact accaaggcta cgatcagtag ctggtctgag 240 aggatgacca gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagtg 300 gggaatcttg cgcaatgggc gaaagcctga cgcagccatg ccgcgtgaat gatgaaggtc 360 ttaggattgt aaaattcttt caccggggac gataatgacg gtacccggag aagaagcccc 420 ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgc tcggaattac 480 tgggcgtaaa gggcgcgtag gcggacattt aagtcagggg tgaaatccca gagctcaact 540 ctggaactgc ctttgatact gggtgtcttg agtgtgagag aggtatgtgg aactccgagt 600 gtagaggtga aattcgtaga tattcggaag aacaccagtg gcgaaggcga catactggct 660 cattactgac gctgaggcgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 720 ccacgccgta aacgatgatt gctagttgtc gggctgcatg cagttcggtg acgcagctaa 780 cgcattaagc aatccgcctg gggagtacgg tcgcaagatt aaaactcaaa ggaattgacg 840 ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc 900 accttttgac atgcctggat cgccacggag acgtggtttt cccttcgggg accaggacac 960 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020 gcgcaaccct cgccattagt tgccatcatt tagttgggaa ctctaatggg actgccggtg 1080 ctaagccgga ggaaggtggg gatgacgtca agtcctcatg gcccttacag ggtgggctac 1140 acacgtgcta caatggcgac tacagagggt taatcccaaa aagtcgtctc agttcggatt 1200 gtcctctgca actcgagggc atgaagttgg aatcgctagt aatcgcggat cagcatgccg 1260 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga gttggttcta 1320 cccgaaggcg atgcgctaac ccgcaaggga ggcagtcgac cacggtaggg tcagcgactg 1380 gggtg 1385 <210> 3 <211> 1460 <212> DNA <213> Ralstonia <400> 3 attgaacgct ggcggcatgc cttacacatg caagtcgaac ggcagcatga tctagcttgc 60 tagattgatg gcgagtggcg aacgggtgag taatacatcg gaacgtgccc tgtagtgggg 120 gataactagt cgaaagatta gctaataccg catacgacct gagggtgaaa gtgggggacc 180 gcaaggcctc atgctatagg agcggccgat gtctgattag ctagttggtg aggtaaaggc 240 tcaccaaggc gacgatcagt agctggtctg agaggacgat cagccacact gggactgaga 300 cacggcccag actcctacgg gaggcagcag tggggaattt tggacaatgg gcgaaagcct 360 gatccagcaa tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact tttgtccgga 420 aagaaatggc tctggttaat acctggggtc gatgacggta ccggaagaat aaggaccggc 480 taactacgtg ccagcagccg cggtaatacg tagggtccaa gcgttaatcg gaattactgg 540 gcgtaaagcg tgcgcaggcg gttgtgcaag accgatgtga aatccccgag cttaacttgg 600 gaattgcatt ggtgactgca cggctagagt gtgtcagagg ggggtagaat tccacgtgta 660 gcagtgaaat gcgtagagat gtggaggaat accgatggcg aaggcagccc cctgggataa 720 cactgacgct catgcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780 cgccctaaac gatgtcaact agttgttggg gattcatttc cttagtaacg tagctaacgc 840 gtgaagttga ccgcctgggg agtacggtcg caagattaaa actcaaagga attgacgggg 900 acccgcacaa gcggtggatg atgtggatta attcgatgca acgcgaaaaa ccttacctac 960 ccttgacatg ccactaacga agcagagatg cattaggtgc tcgaaagaga aagtggacac 1020 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tgtctctagt tgctacgaaa gggcactcta gagagactgc cggtgacaaa 1140 ccggaggaag gtggggatga cgtcaagtcc tcatggccct tatgggtagg gcttcacacg 1200 tcatacaatg gtgcatacag agggttgcca agccgcgagg tggagctaat cccagaaaat 1260 gcatcgtagt ccggatcgta gtctgcaact cgactacgtg aagctggaat cgctagtaat 1320 cgcggatcag catgccgcgg tgaatacgtt cccgggtctt gtacacaccg cccgtcacac 1380 catgggagtg ggctttacca gaagtagtta gcctaaccgc aagggcg attaccacgg 1440 tagggttcat gactggggtg 1460 <210> 4 <211> 1386 <212> DNA <213> Peredibacter <400> 4 aacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gtgaaaaggg gcaaccctta 60 120 agggaaacct gagctaatgc cgcatacgtt agtaataaga aagtgggcgc cgcaagggct 180 catgctgaaa gaagggcctg cggcctatta gctagttggt ggggtaatgg cctaccaagg 240 300 gactcctacg ggaggcagca gtagggaata ttgcgcaatg ggggaaaccc tgacgcagca 360 acgccgcgtg agtgaggaag gtttcggat tgtaaaactc tttttttagg gaaaaatgat 420 tacagagcta ataccctgta aagtgatggt acctaaagaa taagcaccgg ctaacttcgt 480 gccagcagcc gcggtaatac gaagggtgca agcgttgttc ggaatcattg ggcgtaaagc 540 gcgcgcaggc ggatcagcaa gtcagatgtg aaatctcgaa gctcaacttc gaaactgcgt 600 ctgaaactgc tagtctagaa tgtcggaggg ggcagggggaa tttcacgtgt aggggtaaaa 660 tccgtagaga tgtgaaggaa caccggaggc gaaggcgcct gcctggacga ctattgacgc 720 tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgccgtaaa 780 cgatgaatac tagttatgg gggtattgac tccttcagtg acgcagctaa cgcattaagt 840 attccgcctg gggagtacgg tcgcaagact aaaactcaaa caaattgacg ggggcccgca 900 caagcggtgg attatgtggt ttaattcgaa gcaacgcgca gaaccttacc taggcttgaa 960 ctcctacgaa cccgttgtaa tggacggggt gcccgcaagg gaatgtagtg agaggtgctg 1020 catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtctcgcaac gagcgcaacc 1080 cctatcgtct gttgccagca ttaagttggg cactctgacg agactgcctg ggttaaccag 1140 gaggaaggtg gggatgacgt caagtcctca tggcccttat gtctagggct acacacgtaa 1200 tacaatggcg cgtacagagg gaagcgaact cgcaaggggg agcaaatctc aaaaagcgcg 1260 cctcagttcg gattggagtc tgcaactcga ctccatgaag ttggaatcgc tagtaatcgg 1320 agatcagcac gctccggtga atacgttccc gggccttgta cacaccgccc gtcacaccat 1380 gggagt 1386 <210> 5 <211> 1411 <212> DNA <213> Enhydrobacter <400> 5 aacgaacgct ggcggcaggc ttaacacatg caagtcgaac gcgtgtagca atacacgagt 60 ggcgcacggg tgagtaacgc gtggatatct gccttttggt tcggaataac cccgggaaac 120 tggggctaat accggatggt tcctacggga taaagattta tcgccaaaag atgagtccgc 180 gtccgattag ctagttggtg gggtaacggc ctaccaaggc gacgatcggt agctggtctg 240 agaggacgat cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag 300 tgggaatat tggacaatgg gggcaaccct gatccagcca tgccgcgtga gtgatgaagg 360 ccttcggtt gtaaagctct ttggcgggg acgatgatga cggtacccgc agaataagcc 420 ccggctaact tcgtgccagc agccgcggta agacgaaggg ggctagcgtt gttcggaatt 480 actgggcgta aagcgagtgt aggcggttgt tatagtcagg tgtgaaagcc ttgagctca 540 ctcaagaaat gcacttgata ctgaacagct agagtaccgg agaggatagt ggaattccca 600 gtgtagtggt gaatacgta gagattggga agaacaccag tggcgaggc ggctatctgg 660 acggttactg acgctaagac tcgaagcgt ggggagcaaa caggattaga taccctggta 720 gtccacgccg taaacgatgg gtgctagacg ttggcgagct tgctcgtcag tgtcgcagct 780 aacgcgttaa gcaccccgcc tggggagtac ggccgcagg tgaactca aaggaattga 840 cgggggcccg cacaagcggt ggagcatgtg gttcaatcg acgcaacgcg cagaacctta 900 ccagcccttg acatgtgact cgccggctcc agagacggag ccttcggttc ggccggagtc 960 aacacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020 aacgagcgca accctcgtct ccagttgcca tcaggttatg ctgggcactt tggagaaact 1080 gccggtgaca agccggagga aggtggggat gacgtcaagt cctcatggcc cttacgggct 1140 gggctacaca cgtgctacaa tggcggtgac agtgggatgc gaaggggcga cccggagccg 1200 atcccaaaaa gccgtcccag ttcggattgc actctgcaac tcgagtgcat gaaggtggaa 1260 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1320 gccgtcacg ccatgggagt tggttctacc cgaagatggt gcgctaaccg caaggaggca 1380 gccagccacg gtaggatcaa tgactggggc g 1411 <210> 6 <211> 19 <212> DNA <213> Artificial sequence <400> 6 actcctacgg gaggcagca 19 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 ggactachvg ggtwtctaat 20

Claims

1. Use of a detection reagent of a microbial combination for preparing a kit for assisting in the diagnosis of cervical intraepithelial neoplasia (CIN), wherein: The microbial combination includes: Ralstonia and Peredibacter.

2. The use according to claim 1, characterized in that The microbial combination also includes Brevundimonas.

3. The use according to claim 1 or 2, characterized in that The microbial combination also includes enhydrobacter.

4. The use according to claim 1 or 2, characterized in that The microbial combination also includes Pseudomonas.

5. The use according to claim 1, characterized in that The reagents are primers specific to each microorganism, and the primers can detect the 16SrDNA of each microorganism.

6. The use according to claim 1, characterized in that The reagent is a PCR detection reagent.

7. The use according to claim 6, characterized in that The PCR detection reagent includes primers for specifically amplifying each microbial gene.

8. The use according to claim 7, characterized in that The PCR detection reagent also includes a probe that specifically binds to each microbial gene.

9. The use according to claim 1, characterized in that The auxiliary diagnosis comprises the steps of: (1) providing a sample from the cervix of a subject to be tested, and detecting the level of each microorganism in the microbial combination in the sample by a PCR method; (2) Compare the level measured in step (1) with the corresponding microbial level in the normal population.

10. The use according to claim 9, characterized in that In step (1), the 16S rDNA of each microorganism is detected using the PCR method.

Images