Application of intestinal flora marker in functional abdominal pain syndrome of children
Through metagenomic sequencing and bioinformatic analysis of fecal samples of patients with functional abdominal pain syndrome in children and healthy people, Bacteroides common, Rumenococci and Pilori are found to be related to the disease. Develop detection kits and prediction systems for detecting the relative abundance of these bacterial species, solving the difficulties in the diagnosis and treatment of functional abdominal pain syndrome in children, and achieving efficient auxiliary diagnosis and screening of potential treatment options.
Patent Information
- Application Number
- CN202411716611.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-14
AI Technical Summary
The etiology and pathogenesis of functional abdominal pain syndrome in children is not clear. The existing diagnosis and treatment methods need to comprehensively consider a variety of factors, and there is a lack of effective biomarkers for auxiliary diagnosis and treatment.
By collecting fecal samples from children with functional abdominal pain syndrome and healthy people, conducting metagenomic sequencing and bioinformatic analysis, Bacteroides common, Rumenococci and Piloriformis were found to be associated with functional abdominal pain syndrome in children, and detection kits and prediction systems were developed to detect the relative abundance of these bacterial species.
This method can be used as an auxiliary diagnostic tool for children with functional abdominal pain syndrome, has good specificity and sensitivity, and can be used to screen drugs and functional foods for treatment or relief of symptoms.
Smart Images
Figure CN119614688A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the application of intestinal flora markers in the prediction and treatment of functional abdominal pain syndrome in children. Background Art
[0002] Functional abdominal pain syndrome (FAPs), also known as chronic idiopathic abdominal pain or chronic childhood functional abdominal pain syndrome, refers to persistent or frequent episodes of abdominal pain with a course of more than half a year, but is a functional disease that is unrelated or has little to do with the gastrointestinal tract, often accompanied by other systemic discomfort or psychological disorders such as depression and anxiety.
[0003] FAPs are very common in children, with slow onset and abdominal pain. The pain is persistent or recurrent and is not affected by physiological activities (such as diet and defecation). However, its etiology and pathogenesis are not very clear, and may be related to abnormal central pain regulation caused by brain-gut interaction. At present, the prediction, diagnosis and treatment of functional abdominal pain syndrome in children need to consider multiple factors. Existing studies have shown that the intestinal microbiota has become an important factor in the development and persistence of FAPs, and the composition of the intestinal flora changes significantly with age. Summary of the invention
[0004] In view of this, the present invention aims to study the correlation between intestinal microorganisms and functional abdominal pain syndrome in children, in order to provide new ideas for the diagnosis and treatment of functional abdominal pain syndrome in children.
[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides the use of an intestinal flora marker in the preparation of a detection kit for functional abdominal pain syndrome in children, wherein the intestinal flora marker is selected from the prokaryotic organism Bacteroides vulgaris ( Bacteroides vulgatus )、Ruminococcus( Ruminococcus sp. ) and Lachnospirillum ( Lachnospiraceae bacteria ).
[0006] The present invention collects stool samples from patients with functional abdominal pain syndrome in children and healthy people, performs metagenomic sequencing, and uses bioinformatics to perform statistics on sequencing data. Through differential bacteria and linear discriminant analysis, the correlation between common Bacteroides, Ruminococcus and Lachnospira and patients with functional abdominal pain syndrome in children is discovered for the first time; the relative abundance of common Bacteroides, Ruminococcus and Lachnospira in the population with functional abdominal pain syndrome in children is significantly lower than that in the control group (i.e., healthy people). Moreover, the experimental data of the present invention show that the above three strains have good specificity and sensitivity when used as detection variables by ROC curve analysis, so they can be used as detection markers for auxiliary diagnosis of functional abdominal pain syndrome in children.
[0007] Preferably, in the above application, the kit contains reagents for detecting the relative abundance of intestinal flora markers in a fecal sample of a subject.
[0008] The second aspect of the present invention provides a detection kit for functional abdominal pain syndrome in children, which includes reagents for detecting the relative abundance of intestinal flora markers in a subject, including but not limited to microbial genome extraction reagents, sequencing reagents, etc.; wherein the intestinal flora markers are selected from Bacteroides vulgaris, Ruminococcus and Lachnospiraceae.
[0009] The third aspect of the present invention provides the use of intestinal flora markers in establishing a prediction system for functional abdominal pain syndrome in children, wherein the intestinal flora markers are selected from Bacteroides vulgaris, Ruminococcus and Lachnospiraceae.
[0010] Preferably, in the above application, the prediction system uses the relative abundance of intestinal flora markers in the fecal sample of the subject as the detection variable. Moreover, the experimental data of the present invention show that the prediction effect is best when the three bacteria, Bacteroides vulgaris, Ruminococcus and Lachnospirillum, are used in combination.
[0011] A fourth aspect of the present invention provides a prediction system for functional abdominal pain syndrome in children, wherein the prediction system uses the relative abundance of one or more of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae in fecal samples of subjects as detection variables.
[0012] The fifth aspect of the present invention provides the use of intestinal flora markers in screening drugs for treating or alleviating functional abdominal pain syndrome in children, specifically: screening out substances that promote the increase in the relative abundance of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae in the intestine as candidate drugs for treating or alleviating functional abdominal pain syndrome in children.
[0013] The sixth aspect of the present invention provides the use of intestinal flora markers in the preparation or screening of functional foods for alleviating the symptoms of functional abdominal pain syndrome in children. Specifically, the functional food can increase the relative abundance of common Bacteroides, Ruminococcus and Lachnospiraceae in the intestine.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention confirms the correlation between intestinal microorganisms and functional abdominal pain syndrome in children through a study on the association between the composition of intestinal symbiotic flora and functional abdominal pain syndrome in children, and provides markers at the species level that can be used for auxiliary diagnosis and treatment of functional abdominal pain syndrome in children, specifically three species of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae. The combination of these three bacteria has the best effect as a marker for predicting functional abdominal pain syndrome in children, with a sensitivity and specificity of 90.9%.
[0015] Based on the intestinal flora markers mined by the present invention, the present invention further provides a prediction system and method for functional abdominal pain syndrome in children using the relative abundance of intestinal flora markers as detection variables, and experimental data show that it has high specificity and sensitivity. It can be seen that the present invention provides a new approach for the diagnosis of functional abdominal pain syndrome in children.
[0016] The present invention fills the gap in the prior art in the field of intestinal flora spectrum in the diagnosis and treatment of functional abdominal pain syndrome in children, and provides a theoretical basis for developing related detection products, or for alleviating functional abdominal pain syndrome in children through intestinal flora transplantation, or for preparing biological preparations, medicines or functional foods for treating functional abdominal pain syndrome in children. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 A technical roadmap for obtaining a marker of intestinal flora for functional abdominal pain syndrome in children for the present invention; Figure 2 LEfSe score diagram of the three bacterial species in Example 1; Figure 3 It is a box scatter plot of the three strains in Example 1; Figure 4 This is the ROC curve diagram in Example 2. DETAILED DESCRIPTION
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.
[0019] In order to evaluate whether the composition of intestinal symbiotic flora can be used as a detection marker for functional abdominal pain syndrome in children, the present invention collects stool samples from patients with functional abdominal pain syndrome in children and healthy people, performs metagenomic sequencing, and uses bioinformatics to perform statistics on sequencing data to explore intestinal flora associated with functional abdominal pain syndrome in children. Figure 1 As shown, the method for obtaining the intestinal flora marker for children's functional abdominal pain syndrome of the present invention comprises the following steps: (1) Determine the inclusion criteria for patients with functional abdominal pain syndrome and healthy people, and collect stool samples from patients with functional abdominal pain syndrome and healthy people according to the criteria; (2) Extract the microbial genomic DNA from the fecal samples and obtain the original data through metagenomic sequencing; perform quality control on the original data and remove the host sequence to obtain high-quality sequencing data; (3) Analyze the relative abundance of intestinal microbial flora at the species level; (4) Obtain differential bacteria through rank sum test and linear discriminant analysis effect size; (5) Binary logistic regression was used to test the true positive rate and true negative rate of differential bacteria.
[0020] The present invention discovered for the first time the correlation between the prokaryotes Bacteroides vulgaris, Ruminococcus and Lachnospiraceae and patients with functional abdominal pain syndrome in children, and the relative abundance of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae in patients with functional abdominal pain syndrome in children showed a significant decrease; in addition, verification data showed that the ROC curve analysis of the above-mentioned species as detection variables had high specificity and sensitivity, so they can be used as intestinal flora markers in the auxiliary diagnosis and treatment of patients with functional abdominal pain syndrome.
[0021] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.
[0022] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.
[0023] Example 1 This case screened the intestinal flora microorganisms associated with functional abdominal pain syndrome in children, which included the following steps: (1) Sample collection.
[0024] Fecal samples of 43 patients with functional abdominal pain syndrome were collected, and the inclusion criteria included: ① age distribution 0-18 years old; ② patients clinically diagnosed with pediatric functional abdominal pain syndrome; ③ stable vital signs. At the same time, the exclusion criteria of the pediatric functional abdominal pain syndrome group included: ① receiving systemic or local treatment for pediatric functional abdominal pain syndrome one month ago; ② taking antibiotics or probiotics or prebiotics in the past three months; ③ suffering from any chronic disease, including neurobehavioral disorders; ④ receiving medication that affects gastrointestinal motility within 1 week; ⑤ having a history of pain such as functional dyspepsia, aerophagia, abdominal migraine, and functional constipation; ⑥ showing growth disorders; ⑦ suffering from gastrointestinal obstruction or stenosis; ⑧ having a history of abdominal surgery, peptic ulcer or inflammatory bowel disease family history; ⑨ pregnant, planning to become pregnant during the study period, or women of childbearing age who are breastfeeding; ⑩ the researchers believe that the patient is not suitable for inclusion in this study.
[0025] Fecal samples from 43 healthy people were collected as the control group. The inclusion criteria included: ① Age distribution 0-18 years old; ② No diabetes or other metabolic diseases; ③ No depression or other neurological diseases; ④ No irritable bowel syndrome or gastrointestinal diseases; ⑤ No other immune system diseases or not in an immunodeficient state; ⑥ No antibiotics (such as neomycin, rifaximin) or probiotics and prebiotics were taken before and during the study. The exclusion criteria of the control group were the same as those of the children's functional abdominal pain syndrome group.
[0026] The above stool samples and corresponding information were collected from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology, and the specific information is shown in Table 1.
[0027] Table 1 Sample information table used in Example 1
[0028] (2) DNA extraction, library construction and sequencing.
[0029] The CTAB (cetyltrimethylammonium bromide) method was used to extract the microbial genomic DNA of the sample, and the extracted genomic DNA was quality checked to screen out genomic DNA samples with qualified quality. For these selected DNA samples, a library of about 300bp was obtained after random shearing, end repair, connection of A bases, addition of adapters and indexes, and fragment selection. Finally, the inserted fragments were sequenced on the BGI platform using the Paired-End method.
[0030] (3) LEfSe analysis and screening of biomarkers.
[0031] KneadData software was used to perform quality control (based on Trimmomatic) and host removal (based on Bowtie2) on the raw data. Kraken2 was used to calculate the number of sequences of species contained in the sample, and Bracken was used to estimate the actual abundance of species in the sample.
[0032] The abundance data were analyzed using LEfSe software, with the default LDA Score screening value set to 2.5. Figure 2 As shown, three microbial species associated with functional abdominal pain syndrome in children were obtained, namely, Bacteroides common, Ruminococcus and Lachnospiraceae. Moreover, compared with healthy people, the relative abundance of Bacteroides common, Ruminococcus and Lachnospiraceae in stool samples of patients with functional abdominal pain syndrome in children was significantly reduced.
[0033] Example 2 Using the three microorganisms related to children's functional abdominal pain syndrome obtained in Example 1, this example established a functional abdominal pain syndrome prediction system based on intestinal flora, including the following steps: Referring to the standard in step (1) of Example 1, stool samples from 11 children with functional abdominal pain syndrome and 11 healthy subjects were collected from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology in this example. The specific information is shown in Table 2.
[0034] Table 2 Sample information table used in Example 2
[0035] The method in step (2) of Example 1 was used to extract DNA, construct a library and sequence the feces. The same operation as step (3) of Example 1 was then used to perform quality control and analysis on the data to obtain taxonomic abundance data. Specifically, Table 3-4 shows the relevant abundance data of each microbial marker in this example.
[0036] Table 3 Data on the abundance of various microorganisms
[0037] Note: 1 in the type represents healthy people, and 0 represents functional abdominal pain syndrome in children; the combined data of the three bacteria were obtained through loss regression analysis.
[0038] Table 4 Statistical data related to each microorganism
[0039] Note: The mean in the table is the average of relative abundance.
[0040] The above taxonomic abundance data were 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 cutoff value). The specificity and sensitivity calculation and ROC curve drawing were completed using IBM SPSS Statistics (v27) statistical software. The software first calculated the threshold of the actual measurement value, and then calculated 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), and the ROC curve can be constructed by 1-specificity and sensitivity. The integral of the ROC curve is AUC. In order to calculate the specificity and sensitivity of a certain indicator, the Youden coefficient (Youden index = sensitivity + specificity - 1) was first calculated in this example. The specificity and sensitivity corresponding to the maximum value of the Youden coefficient are the specificity and sensitivity of a certain indicator.
[0041] According to the above method, the relative abundance values of microbial markers were used to directly perform ROC curve analysis on the subjects. The ROC diagnostic curves for the prediction scenarios of six bacteria combination, three bacteria combination and single bacteria were as follows: Figure 4 The corresponding AUC, optimal cutoff value, sensitivity and specificity results are shown in Table 5.
[0042] Table 5 ROC diagnostic curve results
[0043] From the above results, it can be seen that when using single bacteria prediction, the predictive ability of common Bacteroides for functional abdominal pain syndrome in children is much higher than that of other bacteria; moreover, the prediction effect of the combination of three bacteria is the best, with an AUC of 0.95, an optimal cutoff value of 0.323, a sensitivity of 90.9%, and a specificity of 90.9%.
[0044] In summary, the present invention discovered for the first time that three bacterial species, namely Bacteroides vulgaris, Ruminococcus and Lachnospiraceae, are associated with functional abdominal pain syndrome in children, and when these three bacterial species are used as predictors of functional abdominal pain syndrome in children, they have good predictive effects. It can be seen that the present invention provides a new approach for the diagnosis and treatment of functional abdominal pain syndrome in children.
[0045] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
Claims
1. The use of intestinal flora markers in the preparation of a detection kit for functional abdominal pain syndrome in children, characterized in that: The intestinal flora marker is selected from Bacteroides vulgaris, Ruminococcus and Lachnospiraceae.
2. The use according to claim 1, characterized in that: The kit contains reagents for detecting the relative abundance of the intestinal flora markers in a fecal sample of a subject.
3. The use according to claim 2, characterized in that: Compared with healthy controls, the relative abundance of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae was reduced in patients with FAPS.
4. A children's functional abdominal pain syndrome detection kit prepared according to any one of claims 1 to 3.
5. Application of intestinal flora markers in establishing a prediction system for functional abdominal pain syndrome in children, characterized in that: The intestinal flora marker is selected from Bacteroides vulgaris, Ruminococcus and Lachnospiraceae.
6. The use according to claim 5, characterized in that: The prediction system uses the relative abundance of each intestinal flora marker in the subject's fecal sample as a detection variable.
7. A prediction system for functional abdominal pain syndrome in children established according to claim 5 or 6.
8. Use of intestinal flora markers in screening drugs for treating or alleviating functional abdominal pain syndrome in children, characterized in that: Screen out substances that increase the relative abundance of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae in the intestine as candidate drugs for treating or alleviating functional abdominal pain syndrome in children.
9. Use of intestinal flora markers in the preparation or screening of functional foods for alleviating symptoms of functional abdominal pain syndrome in children, characterized in that: The functional food can increase the relative abundance of Bacteroides vulgaris, Ruminococcus and Lachnospiraceae in the intestine.