A combination of microbial markers for diagnosing idiopathic membranous nephropathy and its application

Abstract

The present invention belongs to the field of biomedicine and discloses a group of microbial markers for diagnosing idiopathic membranous nephropathy and their applications, and specifically relates to the application of intestinal microbial markers in the diagnosis of idiopathic membranous nephropathy. The present invention, by performing 16S rRNA sequencing on fecal samples of subjects with idiopathic membranous nephropathy, discovered for the first time that 19 types of microbial assemblies are associated with idiopathic membranous nephropathy, suggesting that early diagnosis of idiopathic membranous nephropathy can be achieved by detecting the abundance of 19 types of microorganisms.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and discloses a group of microbial markers for diagnosing idiopathic membranous nephropathy and applications thereof, and particularly relates to the application of intestinal microbial markers in the diagnosis of idiopathic membranous nephropathy. Background Art

[0002] Membranous nephropathy (MN) is a common and intractable glomerular disease characterized by the "three highs and one low" syndrome: severe edema, hyperproteinuria, hyperlipidemia, and hypoproteinemia. Its pathological hallmarks are diffuse glomerular basement membrane (GBM) thickening and immune complex deposition. Recent research indicates a significant upward trend in the incidence of MN, with a younger prevalence. Approximately 80% of cases are renal-limited (idiopathic MN, IMN), while the remaining 20% are related to other systemic diseases or exposures (secondary MN).

[0003] Idiopathic membranous nephropathy (IMN), mentioned here, is a unique, kidney-specific autoimmune glomerular disease. It is the most common cause of nephrotic syndrome in adults and one of the leading identifiable causes of end-stage renal disease (ESRD). Most IMN are mediated by antibodies to the M-type phospholipase A2 receptor (antiphospholipase A2 receptor) (85%), thrombospondin type 1 domain containing 7A (THSD7A) (3%–5%), or other as yet unidentified mechanisms (10%). Currently, early diagnosis of IMN is challenging. In addition to biochemical and immunological markers, renal biopsy and pathological examination of renal tissue, including IgG immunofluorescence testing, are essential for definitive diagnosis. This can cause significant physical trauma and renal damage to the patient. Treatment of IMN primarily involves immunosuppressive therapy, which has significant side effects and a poor prognosis. Effective therapeutic agents and diagnostic and treatment methods are lacking. Therefore, it is urgent to establish a disease diagnostic model for idiopathic membranous nephropathy, which is of great significance for the early detection, early diagnosis and timely treatment of membranous nephropathy.

[0004] The gut microbiome, comprising trillions of bacteria residing in the human gastrointestinal tract, is closely linked to human health. They exist in a symbiotic relationship with the host, performing diverse functions such as regulating immunity, metabolism, host defense, and acting as a biological barrier. Consequently, disturbances in the gut microbiome influence the development of numerous diseases, including obesity, cancer, cardiovascular disease, and kidney disease. Numerous lines of evidence indicate that disturbances in the gut microbiome occur in chronic kidney disease, leading to the proposed gut-kidney axis theory, which better explains the interplay between gut microbes and kidney disease.

[0005] With the application of various technologies and in-depth research, models based on gut microbiome characteristics are increasingly being reported and recognized as diagnostic tools. Individual variability in gut microbiota suggests that gut bacteria have a valuable diagnostic and early warning function. Currently, no gut microbiome-based early diagnostic models for idiopathic membranous nephropathy have been reported, therefore, identifying microbial biomarkers for early diagnosis of idiopathic membranous nephropathy is of great significance. Summary of the Invention

[0006] In order to make up for the shortcomings of the existing technology, the present invention provides an intestinal microbial assembly marker for distinguishing and identifying idiopathic membranous nephropathy. The marker is composed of 19 types of microorganisms. By detecting the abundance levels of 19 types of intestinal microorganisms, early diagnosis of idiopathic membranous nephropathy can be achieved.

[0007] After extensive and in-depth research, the present invention collected stool samples from patients with idiopathic membranous nephropathy and analyzed 16S sequencing results. It was discovered for the first time that the relative abundance of 19 types of microorganisms, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, in idiopathic membranous nephropathy samples was significantly different (P<0.05), indicating that these 19 types of microbial combinations can be used as microbial markers for the early diagnosis of idiopathic membranous nephropathy. The experimental design ideas for discovering this microbial combination are shown in [1]. Figure 1 The basic physiological indicators of all enrolled patients with idiopathic membranous nephropathy and healthy controls are shown in Table 2.

[0008] Table 1. The Chinese names corresponding to the Latin names of 19 bacterial genera.

[0009]

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] One of the technical solutions provided by the present invention is: a group of microbial markers, which are composed of 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter and Citrobacter.

[0012] A second technical solution provided by the present invention is the use of the aforementioned microbial markers in the preparation of a product for diagnosing idiopathic membranous nephropathy. Preferably, the reagent for detecting microorganisms is used in the preparation of a product for diagnosing idiopathic membranous nephropathy, wherein the microorganisms are selected from Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, or Citrobacter.

[0013] Furthermore, the product determines whether the subject has idiopathic membranous nephropathy by measuring the relative abundance of Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter in the sample, and further analyzing and comparing the abundance differences of bacterial genera between the healthy group and the diseased group, wherein the samples include but are not limited to stool samples, intestinal excretions, and intestinal extracts.

[0014] Furthermore, the sample is a stool sample.

[0015] Further, the reagent is selected from:

[0016] Primers that specifically amplify nucleic acid sequences of Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides,, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, Citrobacter, or

[0017] Probes that specifically recognize nucleic acid sequences of Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides,, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, Citrobacter, or

[0018] Antibodies or ligands that specifically bind to protein sequences of Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides,, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, or Citrobacter.

[0019] Furthermore, the primers for specifically amplifying Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter nucleic acid sequences are targeted at the variable regions of 16SrRNA of 19 bacterial genera.

[0020] Furthermore, the disease type is idiopathic membranous nephropathy.

[0021] The present invention provides a system for diagnosing idiopathic membranous nephropathy using microbial markers, comprising:

[0022] Intestinal flora nucleic acid samples are isolated from the test subjects and subjected to high-throughput sequencing to detect the relative abundance of the microbial markers in the intestinal flora. The obtained relative abundance values are analyzed to obtain the critical values of the above-mentioned microbial markers and compare them with the set diagnostic values.

[0023] Furthermore, the microbial markers include Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides,, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter.

[0024] The present invention provides a product for diagnosing idiopathic membranous nephropathy, which includes a reagent for detecting the abundance of 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, in a sample. The product includes a kit, a chip, and a nucleic acid membrane strip.

[0025] Furthermore, the reagents include specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter and Citrobacter.

[0026] Furthermore, the specific primers are primers for detecting 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter 16S rRNA.

[0027] The kit of the present invention includes a reagent for detecting the abundance of Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, and one or more substances selected from the following group: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0028] As an alternative embodiment, the present invention provides a kit for diagnosing nephrotic syndrome based on detecting the abundance of 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter. The kit components are as follows: DNA extraction reagent, primer pairs specifically detecting 16S rRNA of 19 bacterial genera, reaction buffer, deoxynucleotide triphosphates (dNTPs), Taq polymerase reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, and SYBR Green fluorescent dye.

[0029] The kit of the present invention may also be accompanied by an instruction manual for the kit, which describes how to use the kit for testing, and how to use the test results to judge the development of nephrotic syndrome and select a treatment plan.

[0030] The present invention provides the use of 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, in preparing a computational model for predicting idiopathic membranous nephropathy.

[0031] Biomarkers

[0032] The term "biomarker" should be understood in a broad sense, including any detectable biological indicator that can reflect an abnormal state, and can include gene markers, species markers (species markers / genus markers) and functional markers. Among them, the meaning of gene 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, modified DNA or RNA, or unmodified DNA or RNA. In this article, gene markers are sometimes also referred to as characteristic fragments. In particular, the biomarkers of the present invention are microbial markers.

[0033] According to an embodiment of the present invention, stool samples from patients with idiopathic membranous nephropathy are analyzed, and based on 16S rRNA sequencing data, the intestinal flora that exhibit differences in idiopathic membranous nephropathy is statistically analyzed to determine specific sequences associated with idiopathic membranous nephropathy. As a preferred embodiment, the method includes the following steps:

[0034] (1) Sample collection and processing: Collect relevant fecal samples, use a kit to extract DNA, and obtain nucleic acid samples;

[0035] (2) Library construction and sequencing: DNA library construction and sequencing are performed using high-throughput sequencing to obtain the nucleic acid sequences of intestinal microorganisms contained in fecal samples;

[0036] (3) Determine the specific intestinal microbial nucleic acid sequences associated with nephrotic syndrome through bioinformatics analysis methods.

[0037] First, sequencing data of the nucleic acid sequence in the stool sample of the individual is obtained, the sequencing data including multiple reads; the reads are assembled to obtain a gene set, the gene set including multiple assembled fragments, and the assembled fragments in the gene set are non-redundant sequences; the assembled fragments contained in the various microorganisms in the marker are determined; based on the sequencing data, the abundance of each assembled fragment in the gene set is determined, including determining the abundance of the assembled fragments contained in the various microorganisms in the marker; and the abundance of each microorganism is determined based on the determined abundance of the assembled fragments.

[0038] In the present invention, sequencing is performed using a different sequencing platform, including, but not limited to, semiconductor sequencing platforms such as PGM, sequencing-by-synthesis platforms such as Illumina's HISeq and Miseq platforms, and single-molecule real-time sequencing platforms such as the PaCBio platform. Sequencing can be performed using either single-end or paired-end sequencing. The data obtained from the sequencing platform are readout fragments, known as reads.

[0039] In the present invention, the so-called assembly can be performed using known sequence assembly methods or software, such as SOAPdenovo, velvet, etc.

[0040] According to one embodiment of the present invention, by comparing the assembled fragments in the gene set with the microbial reference sequence using the software MetaGeneMark, it is determined whether the assembled fragments are from a certain microorganism based on the degree of similarity with the reference sequence of a certain microorganism. The so-called reference sequence refers to a predetermined sequence, which can be any reference template of the biological category to which the sample to be tested belongs or is contained in advance. For example, if the target is the microorganism in the sample to be tested, the reference sequence can be selected from the reference genomes of various microorganisms in the NCBI database or the DAcc intestinal genome disclosed by the MetaHIT project. Furthermore, a resource library containing more reference sequences can also be pre-configured, for example, based on the status, region, and other factors of the individual from whom the sample to be tested is derived, a closer sequence is selected or determined to be assembled as a reference sequence. According to one embodiment of the present invention, determining the assembled fragments contained in the various microorganisms in the nephrotic syndrome marker includes: comparing the assembled fragments in the gene set with the reference sequences of the various microorganisms, and determining that the assembled fragments with a similarity greater than or equal to 90% with the reference sequence of a microorganism are from the microorganism.

[0041] According to one embodiment of the present invention, in the step of determining the abundance of various microorganisms in the nephrotic syndrome marker based on the abundance of the determined assembly fragments, the abundance of the microorganism is the median or average of the abundance of all assembly fragments contained in the microorganism.

[0042] The term "differential abundance" refers to the presence of elevated or reduced levels of a microorganism in a patient with idiopathic membranous nephropathy compared to normal or control microorganism levels. For the purposes of the present invention, "differential abundance" is defined as a phenomenon occurring when the levels of a microorganism obtained from a normal or diseased subject, or from various stages of a diseased subject, differ by 1.5-fold or greater, approximately 4-fold or greater, approximately 6-fold or greater, or approximately 10-fold or greater.

[0043] In the present invention, the term "sample" includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluids, sputum, alveolar bronchial lavage fluid, urine, feces, etc. Preferably, the sample is tissue or blood. In a specific embodiment of the present invention, the sample is feces.

[0044] In the present invention, "subject" includes animals that can suffer from or develop nephrotic syndrome, and examples of subjects include mammals, for example, humans, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0045] In the present invention, the term "kit" includes an effective amount of reagents for detecting 19 bacterial genera, and one or more substances selected from the group consisting of a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent. Examples include a solution for suspending or fixing cells, a detectable label or marker, a solution for facilitating nucleic acid hybridization, a solution for lysing cells, or a solution for nucleic acid purification.

[0046] The kit of the present invention may also be accompanied by an instruction manual for use of the kit, which describes how to use the kit for detection and how to use the detection results to judge the development of the disease.

[0047] The kit of the present invention can be used to detect 19 bacterial genera using various methods selected from the group consisting of (including but not limited to) real-time quantitative reverse transcription PCR, biochip detection, Southern blotting or in situ hybridization, and immunoassay. Those skilled in the art may adjust and modify the detection method based on actual conditions and needs.

[0048] As a preferred embodiment, the kit detects the abundance of 19 bacterial genera by real-time quantitative reverse transcription PCR; more preferably, the kit includes specific primers for 19 bacterial genera, reaction buffer, deoxynucleotide triphosphates (dNTPs), enzymes such as Taq-polymerase reverse transcriptase, DNase, RNAse inhibitor, DEPC-water, sterile water, etc.

[0049] The term "chip", also known as "array", refers to a solid support comprising connected nucleic acid or peptide probes. An array typically comprises a plurality of different nucleic acid or peptide probes attached to a substrate surface at different known positions. These arrays are also referred to as "microarrays". A "microarray" is an orderly arrangement of hybridization array elements such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies) on a substrate. The substrate can be a solid substrate, for example, a glass or silica slide, beads, fiber optic adhesive, or a semi-solid substrate, such as a nitrocellulose membrane. The nucleotide sequence can be DNA, RNA, or any arrangement thereof.

[0050] The nucleic acid membrane strip of the present invention comprises a substrate and an oligonucleotide probe fixed on the substrate; the substrate can be any substrate suitable for fixing the oligonucleotide probe, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, micro-magnetic beads, etc.

[0051] As used herein, the term "probe" refers to a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe capable of binding to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Probes 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.

[0052] Beneficial effects of the present invention:

[0053] This study, for the first time, discovered that 19 bacterial genera—Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter—are associated with idiopathic membranous nephropathy. The relative abundance of these 19 genera was significantly different between the healthy control group and the diseased group. By detecting the abundance of these 19 genera in subject samples, early diagnosis of idiopathic membranous nephropathy can be achieved. The new microbial biomarker discovered in this study is highly specific, sensitive, and non-invasive, potentially improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A sample study design for discovering intestinal microbiota markers in idiopathic membranous nephropathy

[0055] Figure 2 The abundance of 19 bacterial genera in idiopathic membranous nephropathy samples and healthy control samples is shown in Figure 2.

[0056] Figure 3 Efficacy evaluation of diagnostic models based on microbiome markers DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the examples where specific conditions are not specified are generally carried out under conventional conditions or as recommended by the manufacturer.

[0058] Example 1 Screening of intestinal flora associated with nephrotic syndrome

[0059] 1. Sample collection

[0060] All clinical studies were conducted with the informed consent of the subjects, and the study protocol was approved by the Ethics Committee of Peking Union Medical College. From May to October 2019, a total of 81 subjects were enrolled from Peking Union Medical College Hospital and Peking University Third Hospital, including 40 IMN patients and 41 corresponding healthy controls (see Table 2). All IMN patients eligible for this study were accurately diagnosed as meeting the following criteria: proteinuria, hyperlipidemia, edema, hypoalbuminemia, major immunoglobulin 4 (IgG4) deposition in the glomeruli by immunofluorescence, and platelet-activating protein 2R1 autoantibody (PLA2R1). The exclusion criteria for patients were as follows: (1) patients with complicated diseases, acute and chronic infections; (2) patients with chronic inflammatory bowel disease and celiac disease; (3) patients who received antibiotics, immunosuppressants, and functional foods (probiotics) within three months. Healthy controls had the following characteristics: (1) healthy individuals with normal renal function and no renal disease, celiac disease, or other complications; and (2) patients who had never received antibiotics, immunosuppressants, or functional foods (probiotics) within three months. All participants were aged between 18 and 70 years. The two groups were matched for age and sex.

[0061] Fresh feces were immediately placed in sterile test tubes and stored at -80°C.

[0062] Table 2 Summary of clinical indicators of patients with idiopathic membranous nephropathy and healthy volunteers enrolled in the trial

[0063]

[0064] 2. 16S rRNA sequencing

[0065] The 16S rRNA sequencing of the samples was completed by Beijing Novogene Bioinformatics Technology Co., Ltd. The specific steps are as follows:

[0066] 2.1 Extraction of genomic DNA

[0067] The genomic DNA of the samples was extracted using the CTAB or SDS method, and the operation steps were carried out according to the instructions.

[0068] 1) Sample: Pipette 1000ul of CTAB lysis buffer into a 2.0ml EP tube, add 20ul of lysozyme, add an appropriate amount of sample to the lysis buffer, and incubate at 65°C in a water bath (for fecal samples, the water bath time is 2 hours). Invert and mix several times to ensure that the sample is fully lysed.

[0069] 2) After centrifugation, 950 μl of supernatant was collected and an equal volume of phenol (pH 8.0): chloroform: isoamyl alcohol (25:24:1) was added. The mixture was mixed by inversion and centrifuged at 12,000 rpm for 10 min.

[0070] 3) Remove the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix thoroughly by inversion, and centrifuge at 12,000 rpm for 10 min.

[0071] 4) Pipette the supernatant into a 1.5 mL centrifuge tube, add 3 / 4 volume of isopropanol, shake up and down, and precipitate at -20 degrees.

[0072] 5) Centrifuge at 12,000 rpm for 10 minutes. Pour off the liquid, taking care not to discard the precipitate. Wash twice with 1 ml of 75% ethanol. The remaining small amount of liquid can be collected by centrifugation again and then aspirated with a pipette.

[0073] 6) Blow dry on a clean bench or air dry at room temperature (DNA samples should not be too dry, otherwise they will be difficult to dissolve).

[0074] 7) Add 51 μL ddH2O to dissolve the DNA sample. If necessary, incubate at 55-60°C for 10 minutes to aid dissolution.

[0075] 8) Add 1 μl of RNase A to digest RNA and incubate at 37°C for 15 minutes.

[0076] 2.2 DNA sample purity and concentration determination

[0077] The purity and concentration of DNA were detected by agarose gel electrophoresis. An appropriate amount of sample DNA was placed in a centrifuge tube and diluted with sterile water to 1 ng / μl.

[0078] Detection parameters: Genomic DNA gel concentration: 1%; voltage: 100v; electrophoresis time: 40min; sample volume: 5μL

[0079] 2.3 PCR amplification

[0080] Using diluted genomic DNA as a template, specific primers with barcodes were used according to the selection of sequencing regions. High-Fidelity PCR Master Mix with GC Buffer and high-efficiency, high-fidelity enzymes are used for PCR to ensure amplification efficiency and accuracy.

[0081] 2.3.1 Primer design

[0082] Primer corresponding regions: 16S V3-V4 region primers (341F and 806R): identification of bacterial diversity;

[0083] According to the designated sequencing region, specific primers with barcodes are synthesized. The primer sequences are as follows:

[0084] 341F: 5'-CCTAYGGGRBGCASCAG-3';

[0085] 806R:5'-GGACTACNNNGGGTATCTAAT-3'

[0086] 2.3.2 PCR amplification

[0087] 1) Prepare the reaction system as shown in Table 3.

[0088] Table 3 PCR reaction system

[0089]

[0090] 2) Amplification

[0091] PCR amplification was performed using an Applied Biosystems 2720 PCR instrument. The amplification program was as follows:

[0092] 95°5 min, (94°C1 min, 57°C45 s, 72°C1 min)×35 cycles, 72°C10 min, 16°C5 min.

[0093] 2.4 Mixing and purification of PCR products

[0094] The PCR products were detected by electrophoresis using 2% agarose gel; equal amounts of samples were mixed according to the concentration of the PCR products, and after thorough mixing, the PCR products were detected by electrophoresis using 2% agarose gel, and the target bands were recovered using the gel recovery kit provided by Qiagen.

[0095] Detection parameters: PCR product-gel concentration: 2%; voltage: 80V; electrophoresis time: 40min; sample volume: 3μL

[0096] 2.5 Library construction and sequencing

[0097] use The library was constructed using a DNA PCR-Free Sample Preparation Kit. The constructed library was quantified by Qubit and Q-PCR. Once qualified, it was sequenced using the NovaSeq6000.

[0098] 3 Data Analysis

[0099] 3.1 Sequencing data processing

[0100] According to the barcode sequence and PCR amplification primer sequence, each sample data is split from the offline data. After truncating the barcode and primer sequence, the reads of each sample are spliced using FLASH (V1.2.7, http: / / ccb.jhu.edu / software / FLASH / ). The resulting spliced sequence is the original tag data (Raw Tags); the spliced Raw Tags need to undergo rigorous filtering to obtain high-quality tag data (Clean Tags), referring to the Tag quality control process of Qiime (V1.9.1 http: / / qiime.org / scripts / split_libraries_fastq.html). The tags obtained after the above processing need to be processed to remove chimeric sequences. The tag sequences are compared with the species annotation database (https: / / github.com / torognes / vsearch / ) to detect chimeric sequences, and finally the chimeric sequences are removed to obtain the final effective data (Effective Tags).

[0101] 3.2 OTU clustering and species annotation

[0102] All effective tags from all samples were clustered using Uparse software (Uparse v7.0.1001, http: / / www.drive5.com / uparse / ). Sequences were clustered into OTUs (Operational Taxonomic Units) at a default identity of 97%. Representative sequences from these OTUs were selected based on the algorithm's principles, selecting the most frequently occurring sequence as the representative OTU. OTU sequences were annotated with species using the Mothur method and the SILVA132 (http: / / www.arb-silva.de / ) SSU rRNA database (with a threshold of 0.8–1). Taxonomic information was obtained, and community composition was calculated for each sample at the kingdom, phylum, class, order, family, genus, and species levels. MUSCLE (Version 3.8.31, http: / / www.drive5.com / muscle / ) software was used for rapid multiple sequence alignment to obtain the phylogenetic relationships of all OTU representative sequences. Finally, the data for each sample were normalized.

[0103] 3.3 Analysis of species differences in intestinal flora

[0104] LEfSe (LDA Effect Size) multilevel species differential analysis was used to identify biomarker differences between the two groups. The nonparametric Kruskal-Wallis rank sum test was first used to detect species with significantly different abundances between the groups. Linear discriminant analysis (LDA) was then used to estimate the effect size of each significantly different species. Screening criteria: LDA score > 4 and P value < 0.05.

[0105] 3.4 Random Forest Prediction Model Construction

[0106] To test the diagnostic efficacy of 19 bacterial genera in distinguishing idiopathic membranous nephropathy, 19 bacterial genera were selected to construct a random forest model. The receiver operating characteristic (ROC) curve was used to evaluate the performance of the model, and the area under the curve (AUC) was used to determine the accuracy and specificity of the model.

[0107] 4. Results

[0108] The results are as follows Figure 2 , Figure 3As shown, the abundance of 19 bacterial genera in idiopathic MN samples was significantly different compared with normal controls. The constructed model achieved an AUC of 93.53%, demonstrating that the classification model is highly representative and has excellent diagnostic accuracy. This suggests that the 19-species combination has high specificity, effectively distinguishing idiopathic MN and can be used as a microbial biomarker for the diagnosis of idiopathic MN. This may represent a new approach for disease monitoring and prevention. However, to fully confirm the reliability of the microbial combination, validation in a larger sample size may be necessary.

[0109] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting the abundance of microbial markers in fecal samples in the preparation of a product for diagnosing idiopathic membranous nephropathy, wherein the microbial markers include 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter.

2. The use according to claim 1, characterized in that The products include test kits, chips, and nucleic acid membrane strips.

3. The use according to claim 1, characterized in that The product determines whether a subject has idiopathic membranous nephropathy by measuring the relative abundance of 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, in a sample through reagents or 16S ribosomal sequencing; wherein the relative abundance of the 19 bacterial genera in idiopathic membranous nephropathy is significantly changed compared with that in healthy people, and the significant change means that the relative abundance of the 19 bacterial genera in patients with idiopathic membranous nephropathy is statistically significantly different compared with that in healthy people, that is, the P value is <0.05; the statistical methods include variance test, t-test, and rank sum test.

4. The use according to claim 3, characterized in that The reagent is selected from: Primers that specifically amplify nucleic acid sequences from 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, or Probes that specifically recognize nucleic acid sequences of 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter, or Antibodies or ligands that specifically bind to protein sequences from 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter, and Citrobacter.

5. The use according to claim 4, characterized in that The primers for specifically amplifying the nucleic acid sequences of 19 bacterial genera, including Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter and Citrobacter, are targeted at the variable region of the bacterial genera 16SrRNA.

6. Application of the abundance of microbial markers in fecal samples in constructing a computational model for diagnosing idiopathic membranous nephropathy. The microbial markers include 19 bacterial genera: Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinibacter, Alistipes, unidentified Prevotellaceae, Odoribacter and Citrobacter.

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