A combination of microbial markers related to functional constipation and its application

Abstract

The present invention discloses a combination of microbial markers related to functional constipation and its application. The combination of microbial markers at least includes a specific genus Thomasclavelia of the family Sporobacteraceae, and may also include a specific genus Faecalicatena of the family Lachnospiraceae and / or the genus Eggerthella. By comparing the microbial flora information in the fecal samples of patients with functional constipation and healthy people, the present invention for the first time discovers that these three gut genera are related to functional constipation, and when these three genera are used as detection markers to predict the risk of functional constipation, the prediction method is non-invasive and has a high accuracy rate. The present invention provides new ideas for the prediction and treatment of functional constipation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a combination of microbial markers related to functional constipation and its application in the diagnosis and treatment of functional constipation. Background Art

[0002] Constipation is a common clinical symptom, manifested as dry stools, difficult defecation, and a decrease in the weight and frequency of stools. With the impact of social aging, changes in modern life rhythms and eating habits, and changes in the disease spectrum on diseases, constipation has become one of the important factors affecting the quality of life of modern people. Constipation can be classified into two categories: organic constipation and functional constipation from the perspective of etiology. Organic constipation refers to constipation caused by organic lesions of organs (such as digestive tract diseases, endocrine and metabolic diseases, drug and chemical poisoning, nervous system diseases, etc.), and functional constipation refers to chronic constipation without organic etiology, no structural abnormalities or metabolic disorders, and excluding irritable bowel syndrome. Moreover, the etiology of functional constipation is not yet clear, and its occurrence is related to multiple factors.

[0003] Currently, the commonly used method for diagnosing constipation is for doctors to judge based on symptoms such as whether the patient has not defecated for more than three days, difficult defecation, and the defecation time exceeding half an hour. However, due to the less obvious early symptoms of constipation, patients often tend to ignore them, and for patients who have already shown obvious constipation symptoms, the intestinal mucosa has usually eroded and ulcerated, and there is already inflammation in the colon and rectum, and even intestinal tumors may form. It can be seen that constipation has great harm to the human body, so it is particularly important to detect constipation early and carry out effective treatment. With the increasing attention of people to their own health, there is an urgent need for technologies that can predict constipation in advance.

[0004] More and more evidence has shown that there is a close relationship between the occurrence of constipation and changes in the intestinal flora. In patients with constipation, the microorganisms are dysregulated, so the changes in the flora may be used as an intervention target for constipation or pre - constipation lesions. Summary of the Invention

[0005] In view of this, the present invention aims to obtain microbial markers and methods that can be used to predict functional constipation by studying and comparing the microbial flora information in the fecal samples of patients with functional constipation and healthy people, providing new ideas for the early diagnosis and treatment of functional constipation.

[0006] To achieve the above - mentioned purpose, the technical method of the present invention is as follows:

[0007] In the first aspect of the present invention, a combination of microbial markers related to functional constipation is provided, and the combination of microbial markers includes at least a specific genus of the prokaryotic family Coprobacillaceae Thomasclavelia。

[0008] Preferably, the microbial marker combination may further include at least one of the following two microorganisms: a specific genus of the family Lachnospiraceae Faecalicatena and the genus Eggerthella Eggerthella .

[0009] In the present invention, fecal samples of patients with functional constipation and healthy individuals were collected, and the microbial genomic DNA in the fecal samples was extracted, amplified, library constructed, and metagenomic sequenced. The obtained sequencing data was processed into taxonomic abundance data. Through differential bacteria and linear discriminant analysis using the training set data, three microorganisms with significantly increased abundances in patients with functional constipation were obtained: a specific genus of the prokaryotic family Lachnospiraceae Faecalicatena , the genus Eggerthella Eggerthella , and a specific genus of the family Coprobacillaceae Thomasclavelia , and these three intestinal microorganisms were first discovered in the present invention to be related to constipation; further through the validation set data in the present invention, it was found that when using the ROC curve to analyze the abundances of a specific genus of the prokaryotic family Lachnospiraceae Faecalicatena , the genus Eggerthella Eggerthella , and a specific genus of the family Coprobacillaceae Thomasclavelia as detection variables respectively, they have good specificity and sensitivity, among which the specific genus of the family Coprobacillaceae Thomasclavelia is the best. It can be seen that the intestinal microbial marker combination provided by the present invention can be used as a detection marker for the diagnosis of patients with functional constipation.

[0010] In the context of the present invention, the term "abundance" refers to a measure of the quantity of the target microorganism in a biological sample. The quantification of the abundance of the target nucleic acid sequence in a biological sample may be absolute or relative. "Relative abundance" is usually based on one or more internal reference genes, that is, from the reference strain 16S rRNA gene. For example, using universal primers and expressing the abundance of the target nucleic acid sequence as a percentage of the total bacterial 16S rRNA gene copies or the abundance of the target bacteria determined by normalizing with the Escherichia coli 16S rRNA gene copies. "Absolute abundance" gives the exact number of target molecules by comparison with a DNA standard or by normalizing with the DNA concentration.

[0011] The second aspect of the present invention provides a detection kit for evaluating the risk of functional constipation. Specifically, the detection kit includes reagents for detecting the abundance of a specific genus of the family Coprobacillaceae in the fecal sample of a subject Thomasclavelia , and the abundance of the specific genus of the family Coprobacillaceae Thomasclavelia in the fecal sample of patients with functional constipation is significantly increased.

[0012] Preferably, in the above detection kit, reagents for detecting a specific genus of the family Lachnospiraceae in the fecal sample of a subject may also be includedFaecalicatena and / or Eggerthella Eggerthella reagent for the abundance of, and the specific genus of Faecalicatena Ruminococcaceae Eggerthella and Eggerthella are both significantly increased in the fecal samples of patients with functional constipation.

[0013] Preferably, in the above detection kit, the reagent is a primer for detecting the 16S rRNA corresponding to the specific genus of Thomasclavelia、 the specific genus of Ruminococcaceae Faecalicatena and Eggerthella Eggerthella corresponding to the 16S rRNA.

[0014] The third aspect of the present invention provides a constipation prediction system for evaluating the risk of functional constipation, and the constipation prediction system uses the abundance of the specific genus of Thomasclavelia Coprobacillaceae in the fecal samples of the subject as a detection variable.

[0015] Preferably, in the above constipation prediction system, the detection variable may further include the abundance of the specific genus of Faecalicatena Ruminococcaceae Eggerthella and / or Eggerthella

[0016] in the fecal samples of the subject. Figure 1 The fourth aspect of the present invention provides a method for establishing a constipation prediction system derived from the gut microbiota, specifically as

[0017] shown, including the following steps:

[0018] S1. Determine the inclusion criteria for patients with functional constipation and healthy people;

[0019] S2. Collect fecal samples from patients with functional constipation and healthy people, extract, amplify, construct a library, and perform metagenomic sequencing on the microbial genomic DNA in the fecal samples to obtain raw data;

[0020] S3. Use the same quality control and analysis methods to perform quality control on the raw data and eliminate unqualified data;

[0021] S4. Analyze the relative abundances of the gut microbiota using the same method;

[0022] S5. Screen out the differential bacteria and perform a Wilcoxon rank-sum test and linear discriminant analysis effect size;

[0023] S6. Perform a binary logistic regression test on the true positive rate and true negative rate of the differential bacteria. Thomasclavelia The fifth aspect of the present invention provides the application of the microbial biomarker combination in the preparation of a drug for treating functional constipation, and the microbial biomarker combination includes at least the specific genus of Thomasclavelia, may also include specific genera of the family Lachnospiraceae Faecalicatena and the genus Eggerthella Eggerthella at least one of them, and the drug is used to reduce the relative abundance of the combination of the microbial markers in the intestine.

[0024] The sixth aspect of the present invention provides the application of the above-mentioned combination of microbial markers in screening drugs for functional constipation, specifically: screening out specific genera of the family Coprobacillaceae in the intestine Thomasclavelia , specific genera of the family Lachnospiraceae Faecalicatena and the genus Eggerthella Eggerthella substances with reduced relative abundance as the drug.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] (1) The present invention first discovered specific genera of the family Lachnospiraceae of prokaryotes Faecalicatena , the genus Eggerthella Eggerthella , specific genera of the family Coprobacillaceae Thomasclavelia are related to functional constipation, and these 3 intestinal genera show a significant increase in the population of patients with functional constipation.

[0028] (2) The present invention established a prediction system for evaluating the risk of functional constipation using the above 3 genera of bacteria, verified the prediction possibility of these 3 genera of bacteria, and the results of ROC curve analysis showed that these 3 genera of bacteria had high specificity and sensitivity when used as detection variables, so they could be used as detection markers for the evaluation of the risk of functional constipation, and this detection was completely non-invasive and highly accurate.

[0029] (3) Compared with existing patents such as CN201910101058.8, CN202080093740.8, etc., the present invention used a larger sample size for verification, and the prediction effect was more reliable.

[0030] (4) The present invention provides a means for detecting intestinal flora for patients with functional constipation, provides a bacterial source basis for judging patients with functional constipation and intestinal flora disorders, and provides a basis for later intestinal flora transplantation. Description of the Drawings

[0031] Figure 1 is the technical roadmap of the method for establishing a constipation prediction system derived from intestinal flora in the present invention.

[0032] Figure 2 is the experimental flow chart for screening and verifying microbial markers related to constipation in Example 1 of the present invention.

[0033] Figure 3LEfSe score plots for the three genera in Example 1.

[0034] Figure 4 Box scatter plots for the three genera in Example 1.

[0035] Figure 5 ROC curve graph in Example 1. Detailed implementation manners

[0036] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] Unless otherwise specified, the embodiments are carried out under conventional experimental conditions or according to the conditions recommended in the manufacturer's instructions. The reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0038] Example 1

[0039] In this example, microbial markers capable of being used for the detection of functional constipation were screened and verified, and the experimental process is as Figure 2 shown, and the specific operations are as follows:

[0040] (1) Sample collection.

[0041] Fecal samples of 337 patients with functional constipation and 911 healthy people were collected:

[0042] The samples in the constipation group were from 337 families. The inclusion criteria were: ① Aged 18 - 50 years old; ② Patients diagnosed with constipation; ③ BMI of 18.5 - 25 kg / m 2 . The exclusion criteria were: ① Intestinal constipation caused by congenital factors (such as megacolon) or secondary interventions (such as drugs, endocrine, metabolic, neurological or psychological diseases); ② History of gastrointestinal infection or antibiotic use within the past 1 month; ③ History of previous abdominal surgery, except for cholecystectomy, appendectomy, tubal ligation and cesarean section; ④ History of previous anorectal or perianal surgery; ⑤ Rectal prolapse and / or grade 3 - 4 internal hemorrhoids, in line with the AGA classification; ⑥ Pregnant or lactating women; ⑦ Intestinal pathogen infection; ⑧ Smoking or alcoholism within the past 3 months; ⑨ Uncontrolled liver, kidney, cardiovascular, respiratory or mental diseases; ⑩ Conditions that the researchers considered might affect intestinal transit and microbiota.

[0043] The samples in the control group were from 911 families. The inclusion criteria were: ① Physically healthy without various diseases; ② Age-matched with the patient group (18 - 50 years old). The exclusion criteria were the same as those in the constipation group.

[0044] The above data were from the fecal samples collected by Wuhan Hospital.

[0045] (2) DNA extraction, library construction, and sequencing.

[0046] The CTAB (cetyltrimethylammonium bromide) method was used to extract the microbial genomic DNA of the samples, followed by PCR amplification. The amplification primers were used to construct a library with the TruSeq@DNA PCR-Free Sample Preparation Kit, and then Illumina Miseq PE250 was used for on-machine sequencing.

[0047] (3) Screening biomarkers by LEfSe analysis.

[0048] The downloaded data was filtered, denoised, and spliced to obtain high-quality sequencing data. The Identity standard was set to 100% for clustering to divide the ASV feature sequences. The qiime2 plugin feature-classifier classify-sklearn was used to annotate the feature sequences to generate taxonomic abundance data. Randomly select 80% of the abundance data for analysis using the LEfSe software, and the default screening value of the LDA Score was set to 2.5. The results are as Figure 2 shown. Three specific genera of the Lachnospiraceae family of microorganisms that were significantly enriched in the constipation group were screened out Faecalicatena , Eggerthella Eggerthella , and a specific genus of the Coprobacillaceae Thomasclavelia . The box plots of the three microorganisms are shown in Figure 3 . These three genera were discovered by us for the first time and are related to functional constipation.

[0049] (4) Establishment of a prediction scoring system.

[0050] The remaining 20% of the abundance data was first subjected to binary logistic regression, and then the receiver operating characteristic curve test (ROC curve) analysis was performed to obtain the cutoff value (optimal cut-off value). Among them, the sample information tables involved in steps (3) and (4) are shown in Table 1.

[0051] Table 1 Sample information table

[0052]

[0053] The specificity, sensitivity calculations, and ROC curve plotting were completed using IBM SPSS Statistics (v27) statistical software. The software first calculates the threshold of the actual measured values internally, and then calculates the true positive cases (TP), false positive cases (FP), true negative cases (TN), and false negative cases (FN) corresponding to the threshold. Specificity (true negative rate) = TN / (TN + FP), sensitivity (true positive rate) = TP / (TP + FN). The ROC curve can be constructed through 1 - specificity and sensitivity, and the integral of the ROC curve is the AUC. To calculate the specificity and sensitivity of a certain index, we first calculate the Youden coefficient (Youden index = sensitivity + specificity - 1). The specificity and sensitivity corresponding to the maximum value of the Youden coefficient are the specificity and sensitivity of a certain index.

[0054] The relative abundance values of individual microbial markers were directly subjected to receiver operating characteristic curve test (ROC curve) analysis. Specific genera of Coprobacillaceae Thomasclavelia The AUC of the prediction scoring method was 0.748, the optimal cut-off value was 0.0491, the sensitivity was 61.2%, and the specificity was 86.4%. The ROC curve of the prediction score is as Figure 5 shown. The AUC, optimal cut-off value, sensitivity, and specificity of the prediction scoring methods for 3 genera are shown in Table 2.

[0055] Table 2 Results of ROC diagnostic curve

[0056]

[0057] In summary, the present invention has first discovered that specific genera of the prokaryotic family Lachnospiraceae Faecalicatena , Eggerthella Eggerthella , and specific genera of Coprobacillaceae Thomasclavelia are related to patients with functional constipation, specifically showing significantly higher abundances than those in the healthy population. When using these 3 genera to predict the risk of constipation, the accuracy is high and the prediction method is non-invasive. The present invention provides a new idea for the diagnosis and treatment of patients with functional constipation.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0059] The above embodiments are only for illustrating the technical solutions and features of the present invention, and their purpose is to better enable those familiar with the technology to implement it. It should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention are within the protection scope of the present invention. Those not detailed herein are prior arts.

Claims

1. A combination of microbial markers related to functional constipation, characterized in that, The microbial biomarker combination includes a genus of Thomasclavelia the family Coprobacillaceae, a genus of Faecalicatena the family Lachnospiraceae, and the genus Eggerthella Eggerthella .

2. Use of a reagent for detecting a combination of microbial markers related to functional constipation in the preparation of a detection kit for detecting functional constipation, characterized in that, The reagent is for detecting Thomasclavelia the primers of 16S rRNA of the genus of the family Coprobacillaceae.

3. The use according to claim 2, characterized in that, The reagent further includes a reagent for detecting the abundance of the genera Lachnospiraceae and Eggerthella in a fecal sample of a subject. Faecalicatena and Eggerthella Eggerthella in the fecal sample.

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