Vitiligo detection markers derived from intestinal flora and their applications

Abstract

The present invention discloses a vitiligo detection marker derived from intestinal flora and its application. The vitiligo detection marker includes Blautia, which can be specifically composed of Sutterella, Lachnospira, Parabacteroides, Subdoligranulum, Bifidobacterium and Blautia. The present invention first discovered that these six intestinal bacterial genera are associated with vitiligo patients. When these six bacterial genera are combined as detection markers to diagnose vitiligo, the accuracy rate is high and the diagnosis method is non-invasive. The present invention provides a new idea and approach for the diagnosis and treatment of vitiligo.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a vitiligo detection marker derived from intestinal flora and its application in the diagnosis and treatment of vitiligo. Background Art

[0002] Vitiligo is a common acquired depigmentation skin and mucous membrane disease characterized by depigmentation of the skin and / or hair and a selective reduction or disappearance of melanocytes. To date, the etiology and pathogenesis of vitiligo are still unclear. There are many theories, including autoimmune theory, genetic theory, neurochemical factor theory, oxidative stress theory, melanocyte self-destruction theory, and keratinocyte theory, but no one theory can fundamentally and perfectly explain the cause of vitiligo. Therefore, the prediction, diagnosis, and treatment of vitiligo require comprehensive consideration of multiple factors.

[0003] Intestinal symbiotic flora refers to the large number of microorganisms living in the intestines of animals. With the development of biotechnology, the functions of intestinal microorganisms have been gradually discovered and confirmed, and preparations containing microorganisms are also considered to have great potential in improving human health. Therefore, intestinal symbiotic flora may have unexplored potential in the diagnosis and treatment of vitiligo. Summary of the invention

[0004] In view of this, the present invention aims to provide a new idea and approach for the diagnosis and treatment of vitiligo by exploring the relationship between vitiligo and the composition of intestinal symbiotic flora. In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: In the first aspect of the present invention, the application of intestinal flora microorganisms as vitiligo detection markers is provided, wherein the vitiligo detection markers include Blautia spp. Blautia .

[0005] Preferably, in the above application, the vitiligo detection marker is a prokaryotic organism of the genus Sutterella Sutterella , Lachnospira Lachnospira , Parabacteroides Parabacteroides , rare Micrococcus Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia Blautia composition.

[0006] The present invention collects intestinal samples from vitiligo patients and healthy people, performs 16S rRNA sequencing, and uses bioinformatics to perform statistics on sequencing data, and for the first time discovers the prokaryotic organism Sutterella Sutterella , Lachnospira Lachnospira , Parabacteroides Parabacteroides , rare Micrococcus Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia BlautiaAssociated with vitiligo. Differential bacterial and linear discriminant analysis revealed that Sutterella spp. Sutterella , Lachnospira Lachnospira and Parabacteroides Parabacteroides The abundance of Micrococcus spp. was significantly increased in patients with vitiligo, while the rare Micrococcus spp. Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia Blautia The abundance of Blautia increased significantly in healthy people. Furthermore, the inventors used the abundance level of one or more of the above bacterial genera as detection variables and found through ROC curve analysis that Blautia Blautia And when the six genera are combined, they have high specificity and sensitivity, especially when the six genera are used in combination, the sensitivity and specificity can reach 100%. It can be seen that the six vitiligo-related genera discovered for the first time in the present invention can be used as detection markers to diagnose vitiligo in the test individuals.

[0007] The second aspect of the present invention provides the use of the above-mentioned vitiligo detection marker in the preparation of a product for diagnosing vitiligo, which includes but is not limited to primers, reagents, detection kits, gene chips, diagnostic systems, etc. The product detects the abundance of the vitiligo detection marker in the intestine of the subject and / or evaluates whether the subject is a vitiligo patient or at risk of vitiligo based on the abundance of the vitiligo detection marker.

[0008] Preferably, in the above application, the product for diagnosing vitiligo is a detection kit, comprising a reagent for detecting the abundance of vitiligo detection markers in a fecal sample of a subject, the reagent being capable of analyzing the abundance of the markers by polymerase chain reaction, sequencing, isothermal amplification, bioinformatics or a combination thereof. In some specific embodiments of the present invention, the reagent is a primer for detecting the vitiligo detection marker 16SrRNA.

[0009] Preferably, in the above application, the product for diagnosing vitiligo is a diagnostic system, and the system specifically uses the abundance of vitiligo detection markers in the subject's fecal sample as a detection variable.

[0010] The third aspect of the present invention provides the use of the above-mentioned vitiligo detection marker in the preparation of a drug for alleviating vitiligo-related symptoms.

[0011] The fourth aspect of the present invention provides a method for screening drugs for treating or alleviating vitiligo-related symptoms using the above-mentioned vitiligo detection markers, specifically: screening for drugs that promote the growth of Sutterella in the intestine Sutterella , Lachnospira Lachnospira , Parabacteroides Parabacteroides Reduced abundance and / or promotion of rare Micrococcus species in the gut Subdoligranulum , Bifidobacterium Bifidobacterium、 Blautia BlautiaSubstances with increased abundance as drugs for treating or alleviating the symptoms of vitiligo.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] (1) The present invention explored the correlation between the composition of intestinal symbiotic flora and vitiligo and discovered for the first time the prokaryotic organism Sutterella in the human intestinal flora ( Sutterella ), Lachnospiraceae ( Lachnospira ), Parabacteroides ( Parabacteroides ), rare Micrococcus ( Subdoligranulum ), Bifidobacterium spp. Bifidobacterium ) and Blautia ( Blautia ) and vitiligo patients, among which Sutterella spp. ( Sutterella ), Lachnospiraceae ( Lachnospira ) and Parabacteroides ( Parabacteroides ) in vitiligo patients was significantly higher than that in healthy people, and the rare Micrococcus genus ( Subdoligranulum ), Bifidobacterium spp. ( Bifidobacterium ) and Blautia ( Blautia ) was significantly lower in abundance in vitiligo patients than in healthy controls.

[0014] (2) Based on the six intestinal bacterial genera found to be associated with vitiligo, the present invention further established a method and system for diagnosing vitiligo using the six intestinal bacterial genera as detection variables. The results of ROC curve analysis showed that the six bacterial genera had very high specificity and sensitivity when combined, so they can be used as detection markers for the diagnosis of vitiligo, which is completely non-invasive and highly accurate.

[0015] (3) The present invention fills the gap in the prior art in the research of intestinal flora spectrum in the field of vitiligo technology, and provides a basis for developing related detection products, or for alleviating vitiligo through intestinal flora transplantation, or for preparing biological preparations and drugs for treating vitiligo. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A technical roadmap for exploring the intestinal symbiotic flora associated with vitiligo patients for the present invention.

[0017] Figure 2 This is an experimental flow chart for screening and verifying detection markers for vitiligo diagnosis in Example 1 of the present invention.

[0018] Figure 3 This is the LEfSe score diagram of the six bacterial genera in Example 1.

[0019] Figure 4 This is a box scatter plot of the six bacterial genera in Example 1.

[0020] Figure 5This is the ROC curve diagram in Example 1. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and cannot be used to limit the protection scope of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art; the term "including" and any variations thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0023] The term "abundance" of the present invention refers to a measure of the number of target microorganisms in a biological sample. The quantification of the abundance of a 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, i.e., from a reference strain 16SrRNA gene, such as using universal primers and expressing the abundance of the target nucleic acid sequence as a percentage of total bacterial 16SrRNA gene copies or measured by normalization of Escherichia coli 16SrRNA gene copies.

[0024] In order to evaluate whether the composition of intestinal symbiotic flora can be used as a detection marker for vitiligo, the present invention collects samples from vitiligo patients and healthy people, performs 16SrRNA sequencing, and uses bioinformatics to perform statistics on sequencing data to explore intestinal flora associated with vitiligo. Figure 1 As shown, the present invention specifically obtains intestinal flora microorganisms that can be used as vitiligo detection markers through the following steps:

[0025] (1) Determine the inclusion criteria for vitiligo patients and healthy subjects;

[0026] (2) Fecal samples were collected from vitiligo patients and healthy people, and the microbial genomic DNA in the fecal samples was extracted, amplified, library constructed, and metagenomic sequenced to obtain raw data;

[0027] (3) Use the same quality control and analysis methods to perform quality control on the original data;

[0028] (4) Use the same method to analyze the relative abundance of intestinal microbial flora;

[0029] (5) Screen out differentially expressed bacteria and analyze their effect size using rank sum test and linear discriminant analysis;

[0030] (6) Binary logistic regression was used to test the true positive rate and true negative rate of the differential bacteria.

[0031] The present invention first discovered the prokaryotic organism Sutterella Sutterella , LachnospiraLachnospira , Parabacteroides Parabacteroides , rare Micrococcus Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia Blautia Correlation with vitiligo patients, the training set data was found to be Sutterella spp. through differential bacteria and linear discriminant analysis Sutterella , Lachnospira Lachnospira and Parabacteroides Parabacteroides There was a significant increase in vitiligo patients, while rare Micrococcus spp. Subdoligranulum ), Bifidobacterium spp. ( Bifidobacterium ) and Blautia spp. Blautia The number of bacteria in the healthy population increased significantly, and the validation set data showed that the ROC curve analysis showed that the bacteria had high specificity and sensitivity as detection variables. It can be seen that the above six bacterial genera can be used as detection markers for the diagnosis of vitiligo patients.

[0032] If no specific techniques or conditions are specified in the following examples, the procedures are carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0033] Example 1

[0034] In this case, we screened and verified intestinal flora markers that can be used for the diagnosis of vitiligo. The experimental process is as follows: Figure 2 As shown, the specific operations are as follows:

[0035] (1) Sample collection.

[0036] Fecal samples were collected from 79 patients with vitiligo and 78 healthy subjects (control group):

[0037] The samples of the vitiligo group came from 79 families, and the inclusion criteria were: ① age range 3 to 65 years old; ② patients clinically diagnosed with vitiligo; ③ stable vital signs.

[0038] The exclusion criteria for the vitiligo group were as follows: ① having received systemic or local treatment for vitiligo one month ago; ② having taken systemic antibiotics, systemic hormones, cytokines and immunosuppressants in the past three months; ③ having other autoimmune diseases, gastrointestinal diseases, liver diseases, mental and psychological diseases or other skin diseases; ④ severe, life-threatening diseases such as heart disease, kidney disease, endocrine system diseases, cancer or immunodeficiency diseases; ⑤ women of childbearing age who were pregnant, planned to become pregnant during the study or were breastfeeding; ⑥ the researchers considered the patients unsuitable for inclusion in this study.

[0039] The control group samples came from 78 families, and the inclusion criteria were: ① Age range 3 to 65 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 or prebiotics were taken before and during the study.

[0040] The exclusion criteria of the control group were the same as those of the vitiligo group.

[0041] The above data comes from stool samples collected by Wuhan hospitals.

[0042] (2) DNA extraction, library construction and sequencing.

[0043] The microbial genomic DNA of the samples was extracted using the CTAB (cetyltrimethylammonium bromide) method, followed by PCR amplification. The amplification primers were used to construct the library using the TruSeq@DNA PCR-Free Sample Preparation Kit, and then sequenced using the Illumina Miseq PE250.

[0044] (3) LEfSe analysis and screening of markers.

[0045] The data was filtered, denoised and spliced ​​to obtain high-quality sequencing data. The Identity standard was set to 100% for clustering and ASV feature sequences were divided. The feature sequence was annotated using the qiime2 plug-in feature-classifier classify-sklearn to generate taxonomic abundance data. 80% of the abundance data was randomly selected for analysis using LEfSe software. The default LDA Score screening value was set to 3.5. The results are as follows: Figure 2 As shown. Three microorganisms with significantly increased relative abundance in the vitiligo group were screened out, namely, Sutterella spp. Sutterella , Lachnospira Lachnospira and Parabacteroides Parabacteroides ; and three microorganisms whose relative abundance was significantly reduced in the vitiligo group, namely rare Micrococcus spp. Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia Blautia The box plots of the six bacterial genera are shown in Figure 3 These six bacterial genera were found to be associated with vitiligo for the first time.

[0046] (4) Establishment of a predictive scoring system.

[0047] The remaining 20% ​​abundance data were first subjected to binary logistic regression, and then to receiver operating characteristic curve (ROC) analysis to obtain the cutoff value (optimal cutoff value). The sample information table involved in steps (3) and (4) is shown in Table 1.

[0048] Table 1 Sample information table

[0049]

[0050] 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) is first calculated. The specificity and sensitivity corresponding to the maximum value of the Youden coefficient are the specificity and sensitivity of a certain indicator.

[0051] The receiver operating characteristic curve (ROC) curve analysis was performed based on the relative abundance values ​​of single or multiple microbial markers to obtain the cutoff value (optimal cutoff value). Figure 5 and Table 2), Blautia spp. Blautia ) has a much higher predictive ability for vitiligo than other genera, but the predictive effect of the six bacteria combined is the highest; when the six bacteria are combined, the AUC is 1.000, the optimal cutoff value is 0.500, the sensitivity is 100.0%, and the specificity is 100.0%.

[0052] Table 2 ROC diagnostic curve results

[0053]

[0054] In summary, the present invention first discovered the prokaryotic organism Sutterella Sutterella , Lachnospira Lachnospira , Parabacteroides Parabacteroides , rare Micrococcus Subdoligranulum , Bifidobacterium Bifidobacterium and Blautia Blautia The correlation between the six bacterial genera and vitiligo patients is shown, and when these six bacterial genera are used as detection markers for diagnosing vitiligo, the accuracy rate is high and the diagnosis method is non-invasive; it can be seen that the present invention provides a new idea and approach for the diagnosis and treatment of vitiligo.

[0055] 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. Use of a reagent for detecting the abundance of vitiligo detection markers in a fecal sample of a subject in the preparation of a vitiligo diagnosis product, characterized in that: The vitiligo detection markers are composed of Sutterella, Lachnospira, Parabacteroides, Subdoligranulum, Bifidobacterium and Blautia.

2. The use according to claim 1, characterized in that: The product is a detection kit, which includes reagents for detecting the abundance of vitiligo detection markers in fecal samples of subjects.

3. The use according to claim 1, characterized in that: The product is a diagnostic system that uses the abundance of vitiligo detection markers in the subject's fecal samples as a detection variable.

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