CN114381532A

CN114381532A - Microbial marker combination for diagnosing idiopathic membranous nephropathy and application thereof

Info

Publication number: CN114381532A
Application number: CN202011130811.5A
Authority: CN
Inventors: 张森; 李昭君; 王伟达; 陈园园; 吴海杰
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2022-04-22

Abstract

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

Description

Microbial marker combination for diagnosing idiopathic membranous nephropathy and application thereof

Technical Field

Background

Membranous nephropathy is a clinical multiple refractory glomerular disease, and the clinical prominent manifestations are "three highs and one lowr", namely high edema, high proteinuria, hyperlipidemia, hypoproteinemia. Its pathology is characterized by diffuse Glomerular Basement Membrane (GBM) thickening and immune complex deposition. Relevant research data in recent years show that the incidence rate of membranous nephropathy has a remarkable rising trend and gradually tends to be younger. About 80% of these cases are renal limitation (idiopathic membranous nephropathy, IMN), while the other 20% are associated with other systemic diseases or exposures (secondary membranous nephropathy).

Idiopathic Membranous Nephropathy (IMN) as referred to herein is a unique kidney-specific autoimmune glomerular disease, the most common cause of adult nephrotic syndrome, and one of the major identifiable causes of End Stage Renal Disease (ESRD). Most IMNs are mediated by the M-type phospholipase a2 receptor antibody (anti-phospholipase a2 receptor) (85%), the thrombospondin type 1 domain containing 7A (THSD7A) (3% -5%), or other mechanisms not yet established (10%). At present, the early diagnosis of the idiopathic membranous nephropathy is not very easy, except combining biochemical and immunological indexes, kidney tissue obtained by kidney puncture is very necessary for pathological detection and IgG immunofluorescence diagnosis, and the diagnosis is very necessary, so that the patients can be subjected to larger physical trauma and kidney injury. IMN is mainly treated by immunosuppressant, has large side effect and poor prognosis, and lacks of effective treatment medicaments and diagnosis and treatment means. Therefore, the establishment of disease diagnosis models of idiopathic membranous nephropathy is imminent, which is significant for early detection, early diagnosis and timely treatment of membranous nephropathy.

The trillions of bacteria, intestinal microbiota, present in the human gastrointestinal tract are closely related to human health, and they are in symbiotic relationship with the host, and have various functions of regulating immunity, substance metabolism, host defense, biological barrier, etc. Thus, the disturbance of the intestinal flora affects the development of various diseases, such as obesity, cancer, cardiovascular diseases and kidney diseases. Numerous lines of evidence suggest that intestinal flora is disturbed in chronic kidney disease, and the proposed theory of the gut-renal axis better explains the interaction of intestinal microorganisms with kidney disease.

With the application of various technologies and intensive research, modeling based on intestinal microecological characteristics has been increasingly reported and recognized as a tool for diagnosing diseases. The individual difference of the intestinal microorganisms prompts that the intestinal bacteria have good diagnosis and early warning functions. At present, no intestinal microbial early diagnosis model aiming at the idiopathic membranous nephropathy is reported, so that the search for a microbial marker for the idiopathic membranous nephropathy early diagnosis is of great significance.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention provides an intestinal microorganism combination marker for distinguishing and identifying idiopathic membranous nephropathy, which comprises 19 microorganisms, and can realize early diagnosis of the idiopathic membranous nephropathy by detecting the abundance level of the 19 intestinal microorganisms.

The present invention, through extensive and intensive studies, has found for the first time that 19 microorganisms such as Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabaricteria, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestibacter, Alisistipes, unidentified Prevotella, Odoribacterizer, Citrobacter have a significant difference in relative abundance in samples of idiopathic membranous nephropathy (P <0.05), suggesting that these 19 combinations can be used as early stage biomarkers for the diagnosis of idiopathic membranous nephropathy. The design concept of the test for finding the microbial assembly is shown in FIG. 1; the basic physiological indices of all patients with idiopathic membranous nephropathy and healthy control populations are shown in table 2.

TABLE 1.19 Chinese names corresponding to Latin names of the genera Claudina.

In order to solve the technical problems, the invention adopts the following technical scheme:

one of the technical schemes provided by the invention is as follows: a set of microbial markers consisting of the genera 19 Paraprevotella, Barnesiella, Faecalibacterium, Netivibacterium, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistipes, unidentified votella, Odoribacterium and Citrobacter.

The second technical scheme provided by the invention is as follows: the application of the microbial markers in preparing products for diagnosing idiopathic membranous nephropathy. Use of an agent, preferably for the detection of a microorganism, which is Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Copropacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabaricterioides, Alliosonella, Bacteroides, Butyricicoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevotella, Odoribacter, Citrobacter, for the preparation of a product for the diagnosis of idiopathic membranous nephropathy.

Further, the product determines whether a subject has idiopathic membranous nephropathy by determining the relative abundance of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistips, unidentified Prevolellaceae, Odoribacter, Citrobacter in a sample, including but not limited to fecal samples, intestinal discharges, intestinal extracts.

Further, the sample is a stool sample.

Further, the agent is selected from:

primers which specifically amplify the nucleic acid sequence of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteceae, Odobacter, Citrobacter, or

Probes which specifically recognize the nucleic acid sequence of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteceae, Odobacterbacter, Citrobacter, or

Antibodies or ligands that specifically bind to Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteceae, Odobacter, Citrobacter protein sequences.

Further, the primer specifically amplifying the nucleic acid sequence of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistips, unidentified voPretellaceae, Odoribacter, Citrobacter is directed against the variable region of 16SrRNA of the genus 19.

Further, the disease type is idiopathic membranous nephropathy.

The present invention provides a system for diagnosing idiopathic membranous nephropathy using a microbial marker, comprising:

separating an intestinal flora nucleic acid sample from a detection object, carrying out high-throughput sequencing, detecting the relative abundance of the microbial markers in the intestinal flora, analyzing the obtained relative abundance value to obtain the critical value of the microbial markers, and comparing the critical value with a set diagnosis value.

Further, the microbial markers include Paraprevorella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevolella, Odoribacterizer, Citrobacter.

The invention provides a product for diagnosing idiopathic membranous nephropathy, which comprises reagents for detecting abundances of 19 genera Paraprevotella, Barnesiella, Faecalibacterium, Negativibacillus, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabacteroides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestinebacterium, Alisipes, unidentified Prevotella, Onodobacter, and Citrobacter in a sample, wherein the product comprises a kit, a chip, and a nucleic acid membrane strip.

Further, the reagent includes a primer, a probe, an antisense oligonucleotide, an aptamer or an antibody specific for detecting 19 species of Paraprevorella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Pretevorella, Odoribacter, Citrobacter.

Further, the specific primer is a primer for detecting 19 species of genus Paraprevorella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisisties, unidentified Prevolatecaae, Odoribacter, Citrobacter 16S rRNA.

The kit of the invention comprises reagents for detecting the abundance of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistips, unidentified Prevotella, Odobacter, Citrobacter, and one or more substances selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

As an alternative embodiment, the present invention provides a kit for diagnosing nephrotic syndrome based on the detection of the abundance of 19 species of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabariidea, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteae, Odoribacter, Citrobacter. The kit comprises the following components: DNA extraction reagent, a primer pair for specifically detecting 19-type bacteria 16SrRNA, reaction buffer solution, triphosphate base deoxynucleotides (dNTPs), Taq-polymerase reverse transcriptase, DNase, an RNAse inhibitor, DEPC-water, sterile water and SYBR Green fluorescent dye.

The kit of the present invention may further comprise instructions for use of the kit, wherein the instructions describe how to use the kit for detection, how to use the detection results to determine the progression of nephrotic syndrome, and how to select a treatment regimen.

The present invention provides the use of 19 genera of Paraprevorella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistips, unidentified voPretellaceae, Odoribacter, Citrobacter in the preparation of a computational model for predicting idiopathic membranous nephropathy.

Biomarkers

The term "biomarker" is to be understood in a broad sense and includes any detectable biomarker capable of reflecting an abnormal condition and may include genetic markers, species markers (species/genus markers) and functional markers. The meaning of the gene marker is not limited to the existing gene that can be expressed as a biologically active protein, and includes any nucleic acid fragment, which may be DNA, RNA, modified DNA or RNA, or unmodified DNA or RNA. Genetic markers may also sometimes be referred to herein as signature fragments. In particular, the biomarkers of the invention are microbial markers.

According to the embodiment of the invention, a fecal sample of the idiopathic membranous nephropathy is analyzed, and the intestinal flora presenting differences in the idiopathic membranous nephropathy is subjected to statistics based on 16SrRNA sequencing data, so that a specific sequence related to the idiopathic membranous nephropathy is determined. As a preferred embodiment, the method comprises the following steps:

(1) collecting and processing samples: collecting related excrement samples, and performing DNA extraction by using the kit to obtain nucleic acid samples;

(2) library construction and sequencing: constructing and sequencing a DNA library by using high-throughput sequencing so as to obtain a nucleic acid sequence of the intestinal microorganisms contained in the fecal sample;

(3) the nucleic acid sequence of the specific intestinal microorganism related to the nephrotic syndrome is determined by the analysis method of bioinformatics.

First, obtaining sequencing data of a nucleic acid sequence in a fecal sample of the individual, the sequencing data comprising a plurality of reads; assembling the reads to obtain a gene set, wherein the gene set comprises a plurality of assembling fragments, and the assembling fragments in the gene set are non-redundant sequences; determining the assembly fragments comprised by each microorganism in the marker; determining the abundance of each assembled fragment in the gene set according to the sequencing data, wherein the abundance of each assembled fragment contained in each microorganism in the marker is determined; determining the abundance of each microorganism according to the determined abundance of the assembled fragments.

In the present invention, sequencing may be selected from, but is not limited to, semiconductor sequencing technology platform such as PGM, sequencing by synthesis technology platform such as HISeq, Miseq sequence platform of Illumina, and single molecule real-time sequencing platform such as pacdio sequence platform, depending on the sequencing platform selected. The sequencing mode can select single-ended sequencing or double-ended sequencing, and the obtained off-line data is a fragment obtained by sequencing and reading, and is called a read segment.

In the present invention, the assembly referred to may be performed using known sequence assembly methods or software, for example using SOAPdenovo, velvet, etc.

According to one embodiment of the present invention, the assembled fragments in the gene set are aligned with a microbial reference sequence using the MetaGeneMark software to determine whether the assembled fragments are from a certain type of microbe based on their similarity to the microbial reference sequence. The reference sequence refers to a predetermined sequence, and may be any reference template of a biological category to which a sample to be tested belongs or which is obtained in advance, for example, if the target is a microorganism in the sample to be tested, the reference sequence may be selected from reference genomes of various microorganisms in the NCBI database and/or DAcc enteric genomes disclosed in the MetaHIT project, and further, a resource library including more reference sequences may be configured in advance, for example, a more similar sequence may be selected or determined to be assembled as the reference sequence according to factors such as the status and region of an individual from which the sample to be tested originates. According to one embodiment of the present invention, determining the assembled fragments comprised by the various microorganisms in the nephrotic syndrome marker comprises: and (3) respectively comparing the assembled fragments in the gene set with the reference sequences of various microorganisms, and determining that the assembled fragments with the similarity of more than or equal to 90 percent of the reference sequences of a microorganism are from the microorganism.

According to an embodiment of the present invention, in the step of determining the abundance of each microorganism in the nephrotic syndrome marker according to the determined abundance of the assembled fragments, the abundance of the microorganism is the median or average of the abundances of all the assembled fragments contained in the microorganism.

The term "abundance difference" refers to a higher or lower level of microorganisms obtained in a patient with idiopathic membranous nephropathy compared to a normal or control in vivo level of microorganisms. For the purposes of the present invention, an "abundance difference" is considered to be a phenomenon that occurs when the level of microorganisms taken from a normal or disease-suffering subject, or from each stage of a disease-suffering subject, differs by 1.5-fold or more, about 4-fold or more, about 6-fold or more, about 10-fold or more.

In the present invention, the term "specimen" includes cells, tissues, organs, body fluids (blood, lymph, etc.), digestive juices, expectoration, bronchoalveolar lavage fluid, urine, feces, etc. Preferably, the sample is tissue or blood. In a particular embodiment of the invention, the sample is feces.

In the present invention, "subject" includes animals capable of suffering from or suffering from nephrotic syndrome, and examples of subjects include mammals, e.g., 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 invention, the subject is a human.

In the present invention, the term "kit" includes a detection effective amount of a reagent for detecting the genus 19, which is one or more selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent. For example, a solution for suspending or immobilizing cells, a detectable label or label, a solution for facilitating hybridization of nucleic acids, a solution for lysing cells, or a solution for nucleic acid purification.

The kit of the present invention may further comprise instructions for use of the kit, wherein the instructions describe how to use the kit for detection and how to use the detection results to determine the development of a disease.

With the kit of the present invention, 19 genera of bacteria can be detected by various methods selected from the group consisting of (including but not limited to): real-time quantitative reverse transcription PCR, biochip detection method, DNA blotting or in situ hybridization method, and immunoassay method. The detection mode can be adjusted and changed by those skilled in the art according to actual conditions and needs.

As a preferred embodiment, the kit detects the abundance of the 19 genus by real-time quantitative reverse transcription PCR; more preferably, the kit comprises a primer specific to the genus Clavibacter 19, a reaction buffer, an enzyme such as deoxynucleotide triphosphate (dNTPs) and Taq-polymerase reverse transcriptase, DNase, an RNAse inhibitor, DEPC-water, sterile water, and the like.

The term "chip", also referred to as an "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays are also known as "microarrays". A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

The nucleic acid membrane strip of the present invention comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

In the present invention, the term "probe" refers to a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The invention has the beneficial effects that:

the invention discovers for the first time that 19 kinds of genera, Paraprevorella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistips, unidentified Prevolella, Odoribacter and Citrobacter, are related to idiopathic membranous nephropathy, and the relative abundance of 19 kinds of genera shows a significant difference between healthy control groups and diseased groups. By detecting the abundance of 19 genera in a subject sample, early diagnosis of idiopathic membranous nephropathy can be achieved. The novel microbial assembly marker discovered by the invention has high specificity, strong sensitivity and no wound, and can improve the life quality of a subject.

Drawings

FIG. 1 is a sample study design for finding markers of intestinal flora for idiopathic membranous nephropathy

FIG. 2 is a graph showing the abundance of 19 genus in samples of idiopathic membranous nephropathy and in samples of healthy control group

FIG. 3 is a potency evaluation of a diagnostic model based on microbial composition markers

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of intestinal flora associated with nephrotic syndrome

1 Collection of samples

All clinical studies were conducted with subjects informed and voluntary, and the study protocol was approved by the ethical committee of the university of medicine, beijing counsel. From 5 months 2019 to 10 months 2019, a total of 81 subjects from the Beijing coordination Hospital and the third Hospital of Beijing university, including 40 IMN patients and 41 corresponding healthy controls, were enrolled (see Table 2). All IMN patients eligible for this study were accurately diagnosed as being eligible for proteinuria, hyperlipidemia, edema, hypoalbuminemia, immunofluorescence detecting major immunoglobulin 4(IgG4) deposition in the glomeruli, and positive for platelet activating protein 2R1 autoantibodies (PLA2R 1). The exclusion criteria for patients were (1) patients with complex disease, acute and chronic infection; (2) chronic inflammatory bowel disease and celiac disease patients; (3) patients who received antibiotics, immunosuppressants and functional foods (probiotics) within three months. The healthy control has the following characteristics that (1) healthy people have normal kidney function and no kidney diseases, abdominal cavity diseases and other complications; (2) patients who never received antibiotics, immunosuppressants and functional foods (probiotics) within three months. All participants were between 18-70 years of age. The two groups were matched in age and gender.

Fresh feces were immediately placed in sterile test tubes and stored at-80 deg.C

TABLE 2 summary of clinical indices of patients with idiopathic membranous nephropathy and healthy volunteers enrolled in the trial

2. 16S rRNA sequencing

The sequencing of the sample 16S rRNA is completed by Beijing Nuo He biogenic bioinformation science and technology company, and the specific steps are as follows:

2.1 extraction of genomic DNA

Extracting the genomic DNA of the sample by using a CTAB or SDS method, and carrying out the operation steps according to the instruction.

1) Sample preparation: absorb 1000ul CTAB lysate to 2.0ml EP tube, add 20ul lysozyme, add right amount of sample to lysate, 65 degrees water bath (for the stool sample, water bath time 2 hours), the period reverse mixing several times, in order to make the sample fully cracked.

2) 950ul of the supernatant was centrifuged, and an equal volume of phenol (pH8.0) was added to the supernatant: chloroform: isoamyl alcohol (25: 24:1), reverse mixing, and centrifuging at 12000rpm for 10 min.

3) The supernatant was taken and an equal volume of chloroform was added: isoamyl alcohol (24:1), reverse mixing, and centrifuging at 12000rpm for 10 min.

4) The supernatant was pipetted into a 1.5mL centrifuge tube and isopropanol was added in an amount of 3/4 volumes to the tube and the tube was shaken up and down to precipitate at-20 ℃.

5) Centrifuge at 12000rpm for 10 minutes and pour out the liquid, taking care not to pour out the pellet. The column was washed 2 times with 1ml of 75% ethanol, and the remaining small amount of liquid was collected by centrifugation again and then aspirated out with a pipette tip.

6) The DNA sample is dried by air or air at room temperature (the DNA sample is not dried too much, otherwise it is difficult to dissolve).

7) Add 51. mu.L of ddH2O to dissolve the DNA sample, and incubate at 55-60 ℃ for 10min to aid dissolution if necessary.

8) RNase A1 ul was added to digest the RNA, and the mixture was left at 37 ℃ for 15 min.

2.2 DNA sample purity and concentration determination

The purity and concentration of DNA are detected by agarose gel electrophoresis, an appropriate amount of sample DNA is taken in a centrifuge tube, and the sample is diluted to 1 ng/. mu.l by sterile water.

Detecting parameters: genomic DNA-gel concentration: 1 percent; voltage: 100 v; electrophoresis time: 40 min; sample loading amount: 5 μ L

2.3 PCR amplification

Using diluted genomic DNA as a template, specific primers with Barcode, New England Biolabs, were used according to the selection of the sequencing region

And carrying out PCR by using a High-Fidelity PCR Master Mix with GC Buffer and High-efficiency and High-Fidelity enzyme to ensure the amplification efficiency and accuracy.

2.3.1 primer design

Primer corresponding region: 16S V3-V4 region primers (341F and 806R): identifying bacterial diversity;

synthesizing specific primers with barcode according to the designated sequencing region, wherein the primer sequences are as follows:

341F：5'-CCTAYGGGRBGCASCAG-3'；

806R:5’-GGACTACNNGGGTATCTAAT-3’

2.3.2 PCR amplification

1) The reaction system was prepared as shown in Table 3.

TABLE 3 PCR reaction System

2) Amplification of

PCR amplification was performed using an Applied Biosystems 2720 PCR instrument, using the following amplification program:

95 ℃ for 5min, (94 ℃ for 1min, 57 ℃ for 45s, 72 ℃ for 1min) x 35 cycles, 72 ℃ for 10min, 16 ℃ for 5 min.

2.4 mixing and purification of PCR products

The PCR product is detected by electrophoresis by using agarose gel with 2 percent concentration; the PCR products were mixed in equal amounts according to the concentrations of the PCR products, the PCR products were detected by 2% agarose gel electrophoresis after being mixed well, and the products were recovered from the target bands using a gel recovery kit supplied by Qiagen.

Detecting parameters: PCR product-gel concentration: 2 percent; voltage: 80 v; electrophoresis time: 40 min; sample loading amount: 3 μ L

2.5 library construction and on-machine sequencing

Use of

The DNA PCR-Free Sample Preparation Kit library construction Kit is used for constructing a library, the constructed library is quantified by Qubit and Q-PCR, and the NovaSeq6000 is used for on-machine sequencing after the library is qualified.

3 data analysis

3.1 sequencing data processing

Splitting each sample data from off-line data according to a Barcode sequence and a PCR amplification primer sequence, splicing reads of each sample by using FLASH (V1.2.7, http:// ccb. jhu. edu/software/FLASH /) after the Barcode and the primer sequence are cut off, and obtaining a spliced sequence which is original Tags data (Raw Tags); the spliced Raw Tags needs to be strictly filtered to obtain high-quality tag data (Clean Tags), and a tag quality control flow of Qiime (V1.9.1http:// Qiime. org/scripts/split _ libraries _ fastq. html) is referred to. The Tags obtained after the treatment needs to be subjected to chimera sequence removal treatment, the Tags sequence is compared with a species annotation database to detect a chimera sequence, and the chimera sequence is finally removed to obtain the final Effective data (Effective Tags).

3.2 OTU clustering and species Annotation

All Effective Tags of all samples are clustered by using Upearse software (Upearse v7.0.1001, http:// www.drive5.com/Uparse /), sequences are clustered into OTUs (operational taxomic units) by default with 97% consistency (Identity), and meanwhile, representative sequences of the OTUs are selected, and the sequences with the highest occurrence frequency in the OTUs are selected as the representative sequences of the OTUs according to the algorithm principle. Species annotation was performed on OTUs sequences, species annotation analysis was performed using the Mothur method with the SSUrRNA database of SILVA132(http:// www.arb-SILVA. de /) (setting threshold values of 0.8-1), and taxonomic information was obtained and used at each taxonomic level: kingdom, phylum, class, order, family, genus, species, and statistics of community composition for each sample. Rapid multiple sequence alignment was performed using MUSCLE (Version 3.8.31, http:// www.drive5.com/MUSCLE /) software to obtain phylogenetic relationships for all OTUs representative sequences. And finally, carrying out homogenization treatment on each sample data.

3.3 species differential analysis of intestinal flora

Performing species difference discrimination analysis between two groups by using LEfSe (LDA Effect size) multilevel species difference discrimination analysis (Biomarker), and detecting species with remarkable abundance difference between different groups by using non-parameter factor Kruskal-Wallis rank sum test; linear Discriminant Analysis (LDA) was then used to estimate the magnitude of the effect for each significantly different species. Screening criteria: LDA score >4 and P value < 0.05.

3.4 random forest prediction model construction

In order to detect the diagnosis efficacy of the 19 genera for distinguishing the idiopathic membranous nephropathy, the 19 genera are selected to construct a random forest model, the performance of the model is evaluated through a Receiver Operating Curve (ROC), and the accuracy and the specificity of the model are determined according to the area under the curve (AUC).

4. Results

The results are shown in fig. 2 and fig. 3, compared with the normal control, the abundance of the 19 types of bacteria in the idiopathic membranous nephropathy sample is significantly different, and the AUC of the constructed model is 93.53%, which indicates that the classification model has good representativeness and extremely high diagnosis accuracy. The combination of 19 microorganisms is suggested to have high specificity, well distinguishes the idiopathic membranous nephropathy and can be used as a microbial marker to be applied to diagnosis of the idiopathic membranous nephropathy. This is perhaps a new approach to monitoring and preventing disease. To truly confirm the reliability of a combination of microorganisms, validation may need to be performed on larger sample data.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A set of microbial markers, wherein said microbial markers comprise the genera 19 Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabaricterioides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alipips, unidentified Prevotella, Odoribacterizer and Citrobacter.

2. Use of a microbial marker according to claim 1 for the preparation of a product for the diagnosis of idiopathic membranous nephropathy.

3. The use of claim 2, wherein the product comprises a kit, a chip, a nucleic acid membrane strip.

4. The use according to claim 2, wherein the product is used to determine the relative abundance of 19 species of Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabaricteria, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alipies, unidentified Prevotella, Odoribacterizer, Citrobacter in a sample by means of reagents or 16S ribosome sequencing to determine whether a subject has idiopathic membranous nephropathy; wherein the relative abundance of the 19 genera in idiopathic membranous nephropathy is significantly altered as compared to a healthy population, said significant alteration being a statistically significant difference in the relative abundance of the 19 genera in idiopathic membranous nephropathy patients as compared to a healthy population, i.e., a P-value < 0.05; the statistical methods include variance test, t-test, rank sum test.

5. The use of claim 4, wherein the sample is tissue, blood, oral saliva, intestinal contents, or feces.

6. The use according to claim 4, wherein the agent is selected from:

primers which specifically amplify a nucleic acid sequence of the 19 genera Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alistipes, unidentified Prevotella, Odoribacter and Citrobacter, or primers for the amplification of a nucleic acid sequence of the 19 genera Paraprevotella, Barnesiella, Faecalibacteriobacterium, Neostilbenaria, Salmonella, and Salmonella

Probes specifically recognizing nucleic acid sequences of the 19 genera Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteae, Odobacter and Citrobacter, or

Antibodies or ligands that specifically bind to 19 species of the genera Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyrimonas, Parabacteroides, Allisonella, Bacteroides, Butyricoccus, Actinomyces, Intestibacter, Alisipes, unidentified Prevoteceae, Odoribacter and Citrobacter protein sequences.

7. The use according to claim 6, wherein the primers specifically amplifying the 19 genera Paraprevotella, Barnesiella, Faecalibacterium, Negatibacter, Coprobacter, Romboutsia, Roseburia, Ralstonia, Butyricimonas, Parabaricterioides, Allisonella, Bacteroides, Butyricicoccus, Actinomyces, Intestibacter, Alithipes, unidentified Prevolellaceae, Odoribacter and Citrobacter nucleic acid sequences are directed against the variable region of the genus 16 SrRNA.

8. A system for predicting idiopathic membranous nephropathy using the microbial marker of claim 1,

separating an intestinal flora nucleic acid sample from a detection object, carrying out high-throughput sequencing, detecting and calculating the relative abundance of the microbial markers in the intestinal flora, analyzing the obtained relative abundance value to obtain the critical value of the microbial markers, and comparing the critical value with a set diagnosis value.

9. Use of the microbial marker of claim 1 in the construction of a computational model for the prognosis of idiopathic membranous nephropathy.