CN113637744B

CN113637744B - Application of microbial marker in judging progress of acute pancreatitis course

Info

Publication number: CN113637744B
Application number: CN202111194865.2A
Authority: CN
Inventors: 吴东; 胡晓敏; 张丁丁; 孙翰; 张晟瑜; 李佳宁; 龚亮; 陈国榕; 宋锴; 范正阳; 杨子涵
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2022-02-08
Anticipated expiration: 2041-10-13
Also published as: CN113637744A

Abstract

The invention discloses application of a microbial marker in judging the progression of the course of acute pancreatitis, wherein the microbial marker is selected from g _ Dialister, g _ Clostridium _ sensu _ strico _1 and/or g _ Eubacterium _ ventriosum _ group, wherein g _ Dialister and g _ Eubacterium _ ventriosum _ group are significantly reduced in MSAP and SAP compared with MAP, g _ Clostridium _ sensu _ strico _1 is significantly increased in MSAP and SAP, and the detection of diagnostic efficacy shows that the combination of the microbial markers, especially the microbial markers, has higher diagnostic efficacy.

Description

Application of microbial marker in judging progress of acute pancreatitis course

Technical Field

The invention belongs to the technical field of biology, and relates to application of a microbial marker in judging the progress of the course of acute pancreatitis.

Background

Acute Pancreatitis (AP) is an acute inflammatory disease of the pancreas, and migration of gallstones into the common bile duct and alcoholism are common causes of AP in adults. The worldwide incidence rate of AP is 4.973.4/10 ten thousand, and the incidence rate is increased in the past 20 years. The incidence of AP in China has increased from 0.19% to 0.71% in the last 20 years (Huangkai hong, Lin Ping, Nie Sheng, etc.. incidence of acute pancreatitis in the last 20 years in Guangdong region and etiology analysis [ J ]. J.2007, 7(3): 140-143.). AP is usually acute, with clinical manifestations mainly including upper abdominal pain, vomiting, fever, increased heart rate, etc., with laboratory examinations showing increased white blood cells, increased blood, urine and ascites amylase, etc., and/or with various degrees of signs of peritonitis. 80% of AP patients have a relatively light degree and self-limiting course, 20-30% of patients are accompanied by local or systemic complications, and the total fatality rate is 5-10% (the digestive disease division of the Chinese medical society, the pancreas disease group, the editorial committee of Chinese pancreas adenopathy, the editorial committee of Chinese digestive magazine. Chinese acute pancreatitis diagnosis and treatment guideline (2013, Shanghai); Chinese digestive magazine. 2013,22(04): 217-222). AP can be classified into Mild Acute Pancreatitis (MAP), moderate acute pancreatitis (MSAP) and Severe Acute Pancreatitis (SAP) according to the severity of the clinical condition. SAP has a high fatality rate of 36-50%, and is extremely high in case of late stage combined infection (Vege SS, Gardner TB, Chari ST, et al, Low mortality and high mortality in segment access microorganisms with out organic failure: a case for reproducing the Atlanta clinical classification to include "modified segment access microorganisms" [ J ]. Am J gateway alcohol 2009,104(3): 710-5.).

SAP accounts for approximately 5% to 10% of AP, characterized by organ failure with persistence (>48 hours). When AP is applied, acinar intracellular trypsinogen is activated to be converted into trypsin, meanwhile elastase, phospholipase A2, complement and kinin bypass are activated simultaneously, then inflammatory cells such as neutrophils, macrophages and the like generate and release a large amount of inflammatory factors I such as interleukin-1 (IL-1), tumor necrosis factor (TNF-a) and the like, and further pathological changes such as vessel wall injury, vascular permeability increase and the like are caused through an oxidative stress mechanism, so that systemic inflammatory response syndrome (systemic inflammatory response syndrome SIRS), multiple organ dysfunction syndrome (multiple organ dysfunction syndrome MDOS), multiple organ failure (multiple organ failure, MOF) and abdominal cavity syndrome (abdominal cavity syndrome DS) and other systemic complications such as acute respiratory distress syndrome (acute respiratory syndrome) organ dysfunction are generated, the fatality rate is obviously increased, and the fatality rate is far higher than that of MSAP.

The development of SAP is largely associated with infection, and studies have shown that about 40% to 70% of SAPs with tissue necrosis are associated with secondary bacterial infection (Uhl W, Warshaw A, Imrie C, et a1.IAP Guidelines for the cosmetic management of the acid microorganisms [ J ]. Pancreatology.2002,2(6): 565. 573). The occurrence and development of SAP are related to the structure and function of the microbial community colonized in the intestinal tract including probiotics, the intestinal microecological balance plays a crucial role, and the definition of the characteristics of the AP intestinal flora has very important significance.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention aims to provide the microbial marker for evaluating the progress of the course of acute pancreatitis and the application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, a microbial marker for progression of acute pancreatitis is selected from one or more of g _ Dialister, g _ Clostridium _ sensu _ stricoto _1, or g _ Eubacterium _ ventriosum _ group.

Further, the microbial marker is selected from any two of g _ Dialister, g _ Clostridium _ sensu _ stricoto _1, or g _ Eubacterium _ ventriosum _ group.

Further, the biomarker is selected from g _ Dialister, g _ Clostridium _ sensu _ stricoto _1, and g _ Eubacterium _ ventriosum _ group.

In a second aspect, the use of a reagent for detecting a microbial marker of the first aspect in a sample for preparing a product for determining the progression of acute pancreatitis is provided.

Further, g _ Dialister and g _ Eubacterium _ ventriosum _ group are significantly down-regulated in MSAP and SAP, and g _ Clostridium _ sensu _ stricoto _1 is significantly up-regulated in MSAP and SAP, compared to MAP.

Further, the reagents include reagents for metagenomic sequencing, 16S sequencing, qPCR sequencing.

Further, the product comprises a kit, a preparation and a chip.

Further, the sample is selected from stool, rectal swab.

Further, the sample is selected from a rectal swab.

In a third aspect, the invention provides a product for judging the progress of the course of acute pancreatitis, wherein the kit comprises a reagent for detecting the microbial marker in the first aspect in a sample.

Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer or an antibody that detects the specificity of the microbial marker.

Further, the specific primer is a primer for amplifying the microbial marker 16 SrRNA.

Further, the product also comprises a reagent for extracting the DNA of the sample.

In a fourth aspect, the invention provides the use of the microbial marker of the first aspect in the construction of a computational model for predicting the progression of the course of acute pancreatitis.

In a fifth aspect, the present invention provides a use of the microbial marker of the first aspect in the preparation of a medicament or food for preventing or treating acute pancreatitis.

The invention has the advantages and beneficial effects that:

the invention discovers for the first time that g _ Dialister, g _ Clostridium _ sensu _ stricoto _1 or g _ Eubacterium _ ventriosum _ group are related to the course of acute pancreatitis, that g _ Dialister and g _ Eubacterium _ ventriosum _ group are significantly down-regulated in MSAP and SAP, and that g _ Clostridium _ sensu _ stricoto _1 is significantly up-regulated in MSAP and SAP. The g _ Dialister, g _ Clostridium _ sensu _ stricoto _1 or g _ Eubacterium _ ventriosum _ group can be used as a detection target for diagnosing and predicting the progress of the acute pancreatitis course.

Drawings

FIG. 1 is a graph of the diagnostic efficacy of g _ Dialister in combination with g _ Clostridium _ sensu _ stricoto _ 1;

FIG. 2 is a graph of the diagnostic performance of the g _ Dialister in combination with the g _ Eubacterium _ ventriosum _ group;

FIG. 3 is a graph of the diagnostic efficacy of g _ Clostridium _ sensu _ stricoto _1 in combination with g _ Eubacterium _ vector _ group;

FIG. 4 is a graph of the diagnostic efficacy of the g _ Dialister in combination with the g _ Clostridium _ sensu _ stric _1 and g _ Eubacterium _ ventriosum _ group.

Detailed Description

The invention firstly finds the correlation between the bacteria and clinical medical indexes of the progression of the acute pancreatitis by taking the crowds of the acute pancreatitis in different progressive stages as objects, and provides a diagnosis technology of the progression of the acute pancreatitis on the basis of the correlation. In order to evaluate whether the microbial flora can be used as a predictor of the progression of the acute pancreatitis course, the microbial flora related to the progression of the acute pancreatitis course is found by collecting rectal cotton swabs of MAP patients and MSAP and SAP patients, comprehensively analyzing the results of 16S rRNA sequencing, metagenomic sequencing and quantitative polymerase chain reaction aiming at specific flora, and the abundance of g _ Dialist, g _ Clostridium _ sensory _ stricoto _1 and g _ Eubacterium _ ventriosum _ group is found to be remarkably different between the MAP patients and the MSAP and SAP patients for the first time by 16S rRNA sequencing, so that g _ Dialist, g _ Clostridium _ sensory _ stricoto _1 or g _ Eubacterium _ ventriosum _ group can be used as a biomarker for the progression diagnosis of the acute pancreatitis course.

When a microbial marker indicates or is a marker for an abnormal process, disease or other condition in an individual, the biomarker is generally described as being high or low in content as compared to the level or value of the microbial marker that indicates or is a marker for a normal process, no disease or other condition in the individual. "increased," "elevated," "upregulated," and any variations thereof are used interchangeably to refer to a value or level of a biomarker in a biological sample that is greater than the value or level (or range of values or levels) of the biomarker that is typically detected in a control individual.

"reduced," "downregulated," and any variations thereof, are used interchangeably to refer to a value or level of a biomarker in a biological sample that is less than the value or level (or range of values or levels) of the biomarker that is typically detected in a control individual.

Furthermore, an increased or decreased biomarker may also be referred to as "differential" or as having a "differential level" or "differential value" as compared to a "normal" level or value of the biomarker that is indicative of, or is a marker for, normal progression or absence of a disease or other condition in an individual. Thus, the "differential abundance" of a biomarker may also be referred to as a variation in the "normal" level of the biomarker.

In an embodiment of the invention, the invention diagnoses the progression of the course of acute pancreatitis by: detecting in a nucleic acid sample from an individual one or more nucleic acid fragments corresponding to a species that is relevant for the diagnosis of acute pancreatitis at an advanced stage of the disease. In particular embodiments, nucleic acid fragments corresponding to g _ Dialister, g _ Clostridium _ sensu _ stricto _1, or g _ Eubacterium _ ventriosum _ group are detected. In practicing the methods described herein, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA are used, and are well known.

In the present invention, the agent for detecting a microorganism or determining the level of a microorganism may be a primer, and the method of sequence amplification using the primer may be, for example, Polymerase Chain Reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), multiplex PCR, touchdown PCR, hot start PCR, nested PCR, PCR amplification, real-time PCR, differential PCR, rapid amplification of cDNA ends, reverse polymerase chain reaction, vector-mediated PCR, thermal asymmetric cross PCR, ligase chain reaction, repair chain reaction, transcription-mediated amplification, autonomous sequence replication, selective amplification reaction of a target base sequence.

In the present invention, the agent for detecting a microorganism or measuring the level of a microorganism may be an antibody, and the corresponding microorganism may be detected or the level of a microorganism may be measured by using an immunological method based on an antigen-antibody reaction. Examples of the Assay method used for this purpose include western blotting, enzyme linked immunosorbent Assay (ELISA), Radioimmunoassay (RIA), radioimmunodification (radioimmunodification), Ouchenkia (Ouchterlony) immunodiffusion, rocket (rocket) immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay (Immunoprecipitation Assay), Complement Fixation Assay (complementary hybridization Assay), Fluorescence Activated Cell Sorter (FACS), and protein chip (protein chip).

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "diagnosis" as used herein refers to the differentiation or determination of a disease, syndrome or condition, or to the differentiation or determination of a person having a particular disease, syndrome or condition. In an illustrative embodiment of the invention, the course of acute pancreatitis in a subject is diagnosed based on the analysis of a flora marker in a sample.

The term "fragment" as used herein means a polynucleotide of at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 1000 nucleotides or more in length.

The term "nucleic acid" as used herein broadly refers to: a segment of a chromosome; fragments or portions of DNA, cDNA and/or RNA. Nucleic acids can be obtained or obtained from a nucleic acid sample that is initially separated from any source (e.g., isolated from, purified from, amplified from, cloned or reverse transcribed from sample DNA or RNA).

The term "oligonucleotide" as used herein denotes a short polymer composed of deoxyribonucleotides, ribonucleotides, or any combination thereof. The length of the oligonucleotide is typically between 10 nucleotides and about 100 nucleotides in length. The oligonucleotide is preferably from 15 nucleotides to 70 nucleotides in length, most typically from 20 nucleotides to 26 nucleotides. Oligonucleotides may be used as primers or probes.

An oligonucleotide is "specific" for a nucleic acid if, when the oligonucleotide and the nucleic acid are aligned, the oligonucleotide has at least 50% sequence homology with a portion of the nucleic acid. Oligonucleotides specific for a nucleic acid are those which: under suitable hybridization or wash conditions, it is capable of hybridizing to a target of interest and does not substantially hybridize to nucleic acids not of interest. Higher degrees of sequence homology are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence homology.

The term "hybridize" or "specifically hybridize" as used herein refers to the annealing of two complementary nucleic acid strands to each other under conditions of appropriate stringency. Hybridization is generally carried out using nucleic acid molecules of probe length. Nucleic acid hybridization techniques are well known in the art. Those skilled in the art know how to estimate and adjust the stringency of hybridization conditions such that sequences with at least the desired degree of complementarity will stably hybridize, while sequences with lower complementarity will not stably hybridize.

The term "amplification" as used herein denotes one or more methods known in the art for replicating a target nucleic acid and thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. The target nucleic acid may be DNA or RNA. The sequences amplified in this way form "amplicons". Although the exemplary methods described below involve amplification using the polymerase chain reaction ("PCR"), many other methods for amplifying nucleic acids are known in the art (e.g., isothermal methods, rolling circle methods, etc.). Those skilled in the art will appreciate that these other methods may be used in place of or in addition to the PCR method.

The term "target nucleic acid" or "target nucleotide" as used herein refers to a fragment of a chromosome for which a probe or primer is designed, a complete gene with or without intergenic sequences, a fragment or portion of a gene with or without intergenic sequences, or a nucleic acid sequence. The target nucleic acid may include: a wild-type sequence; a nucleic acid sequence comprising a mutation, deletion or duplication; repeating in series; a gene of interest; a region of a gene of interest or any upstream or downstream region thereof. The target nucleic acid may represent an alternative sequence or allele to a particular gene. The target nucleic acid may be obtained from genomic DNA, cDNA or RNA. The target nucleic acid used herein may be a natural DNA or a PCR-amplified product. In one embodiment, the target nucleic acid is a fragment of a 16S ribosomal RNA gene from a bacterial population.

The term "sample" or "test sample" as used herein refers to any liquid or solid material containing nucleic acids. In suitable embodiments, the test sample is obtained from a biological source (i.e., a "biological sample"), such as cells in culture, or is a tissue sample from an animal, and most preferably from a human. In an exemplary embodiment, the sample is a rectal swab.

The methods and compositions of the present invention can be used to detect nucleic acids associated with various bacteria using a biological sample obtained from an individual. The nucleic acid (DNA or RNA) may be isolated from the sample according to any method known to those skilled in the art. The biological sample may be obtained by standard procedures and may be used immediately or may be stored for later use under conditions appropriate for that type of biological sample.

The starting material for the detection assay is typically a clinical specimen suspected of including g _ Dialister, g _ Clostridium _ sensu _ stricoto _1, or g _ Eubacterium _ ventriosum _ group. The nucleic acids can then be separated from the proteins and carbohydrates present in the original sample. Any purification method known in the art may be used in the context of the present invention. The nucleic acid sequences in the sample can be successfully amplified using in vitro amplification, such as PCR. Generally, any compound that inhibits a polymerase can be removed from the nucleic acid. 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 methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions.

Example 1 detection of microbial flora associated with acute pancreatitis

1. Collection of samples

20 rectal cotton swab samples for Mild Acute Pancreatitis (MAP), 40 rectal cotton swab samples for Moderate Severe Acute Pancreatitis (MSAP) and Severe Acute Pancreatitis (SAP) were collected.

Rectal cotton swab samples from 20 healthy persons and 60 patients with acute pancreatitis. The patient profile statistics are shown in table 1.

Inclusion criteria for patients: meets the classification standard of acute pancreatitis of New Atlanta in 2012.

Exclusion criteria for patients: absence of immunodeficiency, allergy, asthma, colon cancer, diabetes, HIV, inflammatory bowel disease, irritable bowel syndrome, gastroenteritis, narcotic enterocolitis, and arthritis. Antibiotics, probiotics, herbal medicines and other substances which may affect the structure of the intestinal flora are taken.

TABLE 1 patient data

2. 16S rRNA sequencing

2.1 extraction of DNA

Bacterial DNA was extracted from the samples using a DNA extraction kit and the procedure was as described.

2.2 DNA sample purity and concentration determination

Genomic DNA was detected by electrophoresis on a 1% agarose gel.

2.3 PCR amplification and product purification

Synthesizing specific primers with barcode or synthesizing fusion primers with staggered bases according to the designated sequencing region.

In order to ensure the accuracy and reliability of subsequent data analysis, two conditions need to be met, 1) low-cycle amplification is used as far as possible; 2) ensure that the amplification cycles of each sample are consistent. Representative samples were randomly selected for pre-experiments to ensure that the majority of samples were able to amplify the appropriate concentration of product at the lowest cycle number.

PCR was performed using a TransGen AP 221-02: TransStart Fastpfu DNA Polymerase;

a PCR instrument: ABI GeneAmp 9700 type;

all samples are carried out according to formal experimental conditions, each sample is repeated for 3 times, PCR products of the same sample are mixed and detected by 2% agarose gel electrophoresis, the PCR products are recovered by cutting gel by using an AxyPrepDNA gel recovery kit (AXYGEN company), and Tris-HCl is eluted; and (5) detecting by 2% agarose electrophoresis.

2.4 fluorescent quantitation

The PCR product was detected and quantified using a QuantiFluor-ST blue fluorescence quantification system (Promega corporation), and then mixed in the corresponding ratio according to the sequencing amount of each sample.

2.5 Miseq library construction

1) Adding an Illumina official adaptor sequence to the outer end of the target region by PCR;

2) cutting gel by using a gel recovery kit to recover a PCR product;

3) eluting with Tris-HCl buffer solution, and detecting by 2% agarose electrophoresis;

4) sodium hydroxide denaturation produces single-stranded DNA fragments.

Reagent: TruSeqTM DNA Sample Prep Kit

2.6 Miseq sequencing

1) One end of the DNA fragment is complementary with the basic group of the primer and is fixed on the chip;

2) using the DNA fragment as a template and a base sequence fixed on the chip as a primer to carry out PCR synthesis, and synthesizing a target DNA fragment to be detected on the chip;

3) after denaturation and annealing, the other end of the DNA fragment on the chip was randomly complementary to another primer in the vicinity and also immobilized to form a "bridge";

4) performing PCR amplification to generate a DNA cluster;

5) the DNA amplicon is linearized into a single strand;

6) adding modified DNA polymerase and 4 kinds of fluorescence labeled dNTPs, and synthesizing only one base in each cycle;

7) scanning the surface of the reaction plate by laser, and reading the nucleotide species polymerized by the first round of reaction of each template sequence;

8) chemically cleaving the "fluorophore" and the "stop group" to restore the 3' terminal viscosity and continuing to polymerize a second nucleotide;

9) and counting the fluorescent signal result collected in each round to obtain the sequence of the template DNA fragment.

3. Data analysis

3.1 data preprocessing

MiSeq sequencing obtains double-end sequence data, firstly, according to the overlap relation between PE reads, pairs of reads are spliced (merge) into a sequence, meanwhile, quality control filtration is carried out on the quality of the reads and the effect of the merge, samples are distinguished according to barcode and primer sequences at the head end and the tail end of the sequence to obtain an effective sequence, and the sequence direction is corrected to obtain optimized data.

Data culling method and parameters:

1) filtering bases with tail mass value of less than 20 of reads, setting a window of 50bp, if the average mass value in the window is less than 20, cutting back-end bases from the window, filtering reads with quality control of less than 50bp, and removing reads containing N bases;

2) according to the overlap relation between PE reads, splicing (merge) pairs of reads into a sequence, wherein the minimum overlap length is 10 bp;

3) the maximum mismatch ratio allowed by the overlap region of the splicing sequence is 0.2, and non-conforming sequences are screened;

4) distinguishing samples according to the barcode and the primers at the head end and the tail end of the sequence, and adjusting the sequence direction, wherein the number of mismatch allowed by the barcode is 0, and the maximum number of mismatch of the primers is 2;

using software: FLASH and trimmatic.

3.2 species annotation and evaluation

The OTU clustering analysis is carried out by using Usearch, and OTU (operational Taxonomic units) are unified marks artificially set for a certain classification unit (strain, genus, species, grouping and the like) in phylogenetic science or population genetics research for facilitating analysis. To know the number information of species, genus, etc. in the sequencing result of a sample, the sequence needs to be clustered (cluster). Through clustering, sequences are classified into a plurality of groups according to the similarity of the sequences to each other, and one group is an OTU. OTUs partitioning can be performed for all sequences according to different similarity levels, typically with bioinformatic analysis of OTUs at 97% similarity level.

A software platform: usearch (vsesion 7.0 http:// drive5.com/uparse /)

The analysis steps are as follows:

non-repetitive sequences are extracted from the optimized sequences, so that the redundant calculation amount in the middle process of analysis is reduced conveniently;

removing non-repeated single sequences;

OTU clustering was performed on non-repeated sequences (containing no single sequence) according to 97% similarity, and chimeras were removed during clustering to obtain representative sequences of OTUs.

Performing taxonomic analysis on OTU representative sequences with 97% similarity level by using RDP classifier Bayesian algorithm, and performing taxonomic analysis at each taxonomic level: domain, kingdom, phylum, class, order, family, genus, and species.

3.3 species Difference analysis

Species differential analysis is analyzed by bioinformatics analysis methods to detect differences in abundance exhibited by different groups (or samples) of microbial communities based on the obtained community abundance data. The content of species differential analysis includes: and (3) carrying out difference significance test between groups and Lefse multi-level species difference discriminant analysis. This project used the significance test of differences between groups to screen for different species.

Significance test of differences between groups species exhibiting abundance differences among different groups (samples) of microbial communities can be detected using rigorous statistical methods based on the obtained community abundance data, and a hypothesis test is performed to assess the significance of the observed differences. The analysis can select different classification levels of domains, kingdoms, phyla, classes, orders, families, genera, species, OTU, etc.

1) The Wilcox rank-sum test, also known as the Mann-Whitney U test, is a method of nonparametric testing of two independent sets of samples. The original assumption is that two populations of independent samples have no significant difference in distribution, and the average ranks of the two populations of samples are researched to judge whether the two populations of samples have difference in distribution, so that the analysis can be used for performing significant difference analysis on the species of the two populations of samples and correcting the P value by various methods.

2) The multiple test correction, i.e. the multiple test correction method for P value is "fdr".

3) And a two-tailed test for specifying the type of confidence interval to be evaluated, and selecting the two-tailed test (confidence interval).

4) A CI calculation method, i.e., a method of calculating a confidence interval, the method being DP: welch's confidence updated. Selecting confidence: 0.95.

calculating the influence size (effect size) by using a DP method, namely mean1-mean 2; confidence intervals were calculated using the Welch T test and the screening criteria P < 0.05.

Software: the stats package of R and the scipy package of python.

4. Results

The results show that g _ Dialister and g _ Eubacterium _ ventriosum _ group are significantly down-regulated in the SAP and MSAP groups compared to MAP; g _ Clostridium _ sensu _ stricoto _1 was significantly upregulated in the SAP and MSAP groups, the difference was statistically significant (P < 0.05), suggesting that g _ Dialister, g _ Clostridium _ sensu _ stricoto _1, or g _ Eubacterium _ ventriosum _ group could be used as biomarkers for the diagnosis of progression of the course of acute pancreatitis.

Example 2 detection of diagnostic potency of microbial markers

The sensitivity and specificity of g _ Dialister, g _ Clostridium _ sensu _ stricoto _1 and/or g _ Eubacterium _ ventriosum _ group for acute pancreatitis diagnosis were analyzed by plotting a subject working characteristic curve (ROC) using R according to the abundance of g _ Dialister, g _ Clostridium _ sensu _ stricoto _1 or g _ Eubacterium _ ventriosum _ group.

The ROC curve and the characteristic parameters are shown in fig. 1-4 and table 2, respectively, and the area under the curve with g _ Dialister as the detection index is 0.674, the area under the curve with g _ Clostridium _ sensu _ stricoto _1 as the detection index is 0.667, and the area under the curve with g _ Eubacterium _ ventriosum _ group as the detection index is 0.666.

The area under the curve of the g _ Dialister in combination with g _ Clostridium _ sensu _ stricoto _1 was 0.785, and the specificity and sensitivity under the optimal threshold calculation were 0.800, 0.650, respectively (fig. 1); the area under the curve for g _ Dialister in combination with g _ Eubacterium _ ventriosum _ group was 0.755, and the specificity and sensitivity under the optimal threshold calculation were 0.800, 0.625, respectively (fig. 2); the area under the curve for g _ Clostridium _ sensu _ stricoto _1 in combination with g _ Eubacterium _ ventriosum _ group was 0.831, with specificity and sensitivity at optimal threshold calculation of 0.850, 0.725, respectively (fig. 3); the area under the curve for the g _ Dialister in combination with the g _ Clostridium _ sensu _ stricoto _ and g _ Eubacterium _ ventriosum _ group is 0.902, and the specificity and sensitivity under optimal threshold calculation are 0.950, 0.775, respectively (fig. 4). The application of the g _ Dialister, g _ Clostridium _ sensu _ stric _1 or g _ Eubacterium _ ventriosum _ group, especially the combination thereof, in the diagnosis or prediction of the progress of the acute pancreatitis course is shown to have higher accuracy, sensitivity and specificity.

TABLE 2 area under the curve

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. Use of a reagent for detecting a microbial marker in a sample for the manufacture of a product for determining the progression of acute pancreatitis, wherein the microbial marker is a combination of g _ Dialister, g _ Clostridium _ sensu _ stricoto _1 and g _ Eubacterium _ ventriosum _ group.

2. The use of claim 1, wherein the reagents comprise reagents for metagenomic sequencing, 16S sequencing, qPCR sequencing.

3. The use of claim 1, wherein the product comprises a kit, a formulation, a chip.

4. Use according to claim 1, wherein the sample is selected from faeces, rectal swab.

5. The use of claim 1, wherein the agent comprises a primer, probe, antisense oligonucleotide, aptamer or antibody specific for detecting the microbial marker.

6. The use according to claim 5, wherein the specific primer is a primer that amplifies the microbial marker 16 SrRNA.

7. Use according to claim 1, wherein the product further comprises a reagent for extracting the sample DNA.