CN118272552A - Vitiligo detection marker derived from intestinal flora and application thereof - Google Patents
Vitiligo detection marker derived from intestinal flora and application thereof Download PDFInfo
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Abstract
The invention relates to a vitiligo detection marker derived from intestinal flora and application thereof, wherein the vitiligo detection marker comprises Blauthia of BlueTorili, and can be specifically composed of BlueTorili Sutterella, lachnospira, paralopecuroides Parabacteroides, pediococcus Subdoligranulum, bifidobacterium and BlueTorili. The invention discovers that the 6 kinds of intestinal bacteria are related to vitiligo patients for the first time, and when the 6 kinds of bacteria are combined to be used as detection markers for diagnosing vitiligo, the accuracy is high, the diagnosis mode is noninvasive, and the invention provides a new thought and approach for the diagnosis and treatment of vitiligo.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a vitiligo detection marker derived from intestinal flora and application thereof in diagnosis and treatment of vitiligo.
Background
Vitiligo is a common acquired pigment-depriving skin mucosa disease characterized by pigment deprivation and melanocyte selectivity reduction or disappearance of skin and/or hair. The etiology and pathogenesis of vitiligo are not clear so far, but various theories including autoimmune theory, genetic theory, neurochemical factor theory, oxidative stress theory, melanocyte self-destruction theory and keratinocyte theory are not known, but any theory can not explain the etiology of vitiligo fundamentally and perfectly. Therefore, the prognosis, diagnosis and treatment of vitiligo need to comprehensively consider various factors.
Gut commensal flora refers to a vast number of microorganisms living in the gut of animals. With the development of biotechnology, the functions of intestinal microorganisms are gradually being explored and demonstrated, and preparations containing microorganisms are also considered to have great potential in improving human health. Therefore, the intestinal symbiotic flora may have the potential of not being explored in the aspects of diagnosis, treatment and the like of vitiligo.
Disclosure of Invention
In view of the above, the invention aims to provide a new thought and approach for diagnosis and treatment of vitiligo by exploring the association of vitiligo and intestinal symbiotic flora composition. In order to achieve the above purpose, the technical scheme of the invention is as follows: in a first aspect, the present invention provides the use of a microorganism of the intestinal flora as a marker for the detection of vitiligo, wherein the marker for the detection of vitiligo comprises Blautia of the genus Blautia.
Preferably, in the above application, the vitiligo detection marker consists of procaryote Sagnaporthe Sutterella, chaetoceros Lachnospira, paralopecuroides Parabacteroides, pediococcus Subdoligranulum, bifidobacterium and Blauthia.
According to the invention, through collecting intestinal samples of vitiligo patients and healthy people, carrying out 16SrRNA sequencing and carrying out statistics of sequencing data by using bioinformatics, the correlation of procaryotes Sagnac bacteria Sutterella, chaetoceros Lachnospira, paramycolatopsis Parabacteroides, pediococcus rarefaciens Subdoligranulum, bifidobacterium and Blauthia with vitiligo is discovered for the first time. The differential bacteria and linear discriminant analysis show that Saspecific bacillus Sutterella, maospira Lachnospira and Paramycolatopsis Parabacteroides are obviously increased in the population of vitiligo patients, while the rare micrococcus Subdoligranulum, bifidobacterium Bifidobacterium and Blauthia are obviously increased in the population of healthy people. Furthermore, the inventor uses the abundance level of one genus or a plurality of genus as a detection variable, and finds that Blauthia and 6 genus have higher specificity and sensitivity when being combined through ROC curve analysis, and particularly the sensitivity and specificity can reach 100% when the 6 genus are combined. Therefore, the 6 bacteria related to the vitiligo, which are discovered for the first time, can be used as detection markers for diagnosing the vitiligo of a tested individual.
In a second aspect, the present invention provides the use of the above-mentioned vitiligo detection markers for the preparation of a product for the diagnosis of vitiligo, including but not limited to primers, reagents, detection kits, gene chips, diagnostic systems, etc., for assessing whether a subject is a vitiligo patient or at risk for vitiligo by detecting the abundance of vitiligo detection markers in the intestinal tract of the subject and/or based on the abundance of vitiligo detection markers.
Preferably, in the above application, the product for diagnosing vitiligo is a detection kit comprising reagents for detecting the abundance of a vitiligo detection marker in a fecal sample of a subject, which reagents can analyze the abundance of the marker by polymerase chain reaction, sequencing, isothermal amplification, bioinformatics or a combination thereof. In some embodiments of the invention, the reagent is a primer for detecting the vitiligo detection marker 16 SrRNA.
Preferably, in the above application, the product for diagnosing vitiligo is a diagnostic system, and the system specifically uses abundance of vitiligo detection markers in a fecal sample of a subject as a detection variable.
The third aspect of the invention provides application of the vitiligo detection marker in preparation of medicines or functional foods for relieving vitiligo related symptoms.
The fourth aspect of the present invention provides a method for screening a drug or a functional food for treating or alleviating symptoms associated with vitiligo by using the above-mentioned vitiligo detection markers, which specifically comprises: screening out substances which promote the abundance decrease of Saspecific genus Sutterella, trichosporon Lachnospira and Paramycolatopsis Parabacteroides in intestinal tracts and/or promote the abundance increase of rare micrococcus Subdoligranulum, bifidobacterium and Blaustemperia in intestinal tracts, and using the substances as medicines or functional foods for treating or relieving vitiligo symptoms.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention discovers the relevance of procaryotic Sagnac bacteria (Sutterella), magnaporthe (Lachnospira), paramycolatopsis (Parabacteroides), pediococcus rarefaciens (Subdoligranulum), bifidobacterium (Bifidobacterium) and Blautia with vitiligo patients for the first time by mining the relevance of intestinal symbiotic bacteria composition to vitiligo, wherein the abundance of Sagnac bacteria (Sutterella), magnaporthe (Lachnospira) and Paramycolatopsis (Parabacteroides) in vitiligo patients is significantly higher than healthy people, and the abundance of Pediococcus rarefaciens (Subdoligranulum), bifidobacterium (Bifidobacterium) and Blautia) in vitiligo patients is significantly lower than healthy people.
(2) Based on the found 6 kinds of intestinal bacteria related to the vitiligo, the invention further establishes a method and a system for diagnosing the vitiligo by taking the 6 kinds of intestinal bacteria as detection variables, and the result of ROC curve analysis shows that the 6 kinds of bacteria have very high specificity and sensitivity when combined, so the method can be used as a detection marker for diagnosing the vitiligo, and is completely noninvasive and high in accuracy.
(3) The invention fills the blank of the research of the intestinal flora spectrum in the technical field of vitiligo in the prior art, and provides a basis for developing related detection products, or for alleviating vitiligo through intestinal fungus transplantation, or for preparing biological preparations, medicines or health-care foods for treating vitiligo.
Drawings
FIG. 1 is a technical roadmap for developing intestinal symbiotic flora associated with vitiligo patients according to the present invention.
Fig. 2 is a flow chart of the experiment of screening and verifying the detection markers for vitiligo diagnosis in example 1 of the present invention.
FIG. 3 is a LEfSe score of 6 genus bacteria in example 1.
FIG. 4 is a box-type scattergram of 6 genus bacteria in example 1.
Fig. 5 is a graph of ROC in example 1.
Detailed Description
The following detailed description of the technical solution of the present invention will be given with reference to the accompanying drawings and examples, which are only used to more clearly illustrate the technical solution of the present invention, but are not to be construed as limiting the scope of the present invention.
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 "comprising" and any variations thereof in the description of the invention and in the claims is intended to cover a non-exclusive inclusion.
The term "abundance" in the present invention refers to a measure of the number of target microorganisms in a biological sample. Quantification of abundance of a target nucleic acid sequence within a biological sample may be absolute or relative. "relative abundance" is typically based on one or more internal reference genes, i.e., from a reference strain 16SrRNA gene, such as a sexual bacterium determined using universal primers and expressing the abundance of a target nucleic acid sequence as a percentage of total bacterial 16SrRNA gene copies or normalized by e.coli 16SrRNA gene copies.
In order to evaluate whether the intestinal symbiotic flora composition can be used as a detection marker of vitiligo, the invention discovers the intestinal flora related to vitiligo by collecting samples of vitiligo patients and healthy people, performing 16SrRNA sequencing and counting sequencing data by using bioinformatics. As shown in fig. 1, the present invention specifically obtains intestinal flora microorganisms capable of serving as a marker for detection of vitiligo by:
(1) Determining the grouping standard of vitiligo patients and healthy people;
(2) Collecting fecal samples of vitiligo patients and healthy people, extracting, amplifying, banking and metagenome sequencing microbial genome DNA in the fecal samples, and obtaining raw data;
(3) The same quality control and analysis method is used for controlling the quality of the original data;
(4) Analyzing the relative abundance of intestinal microbial flora by the same method;
(5) Screening out differential bacteria, and analyzing the effect quantity through rank sum test and linear discriminant;
(6) And testing the true positive rate and the true negative rate of the differential bacteria by binary logistic regression.
The invention discovers the relevance of procaryotes Saspecific genus Sutterella, the chaetobacter Lachnospira, the parabacteroides Parabacteroides, the rare micrococcus Subdoligranulum, the Bifidobacterium bifidobactirium and the Blauthentica with vitiligo patients for the first time, training set data show that Saspecific genus Sutterella, the chaetobacter Lachnospira and the parabacteroides Parabacteroides are obviously increased in vitiligo patients through differential bacteria and linear discriminant analysis, and rare micrococcus (Subdoligranulum), the Bifidobacterium (bifidobactirium) and the Blauthentica are obviously increased in healthy people, and further the training set data show that the training set data have higher specificity and sensitivity by using ROC curve analysis as detection variables. Therefore, the 6 bacteria can be used as detection markers for diagnosis of patients with vitiligo.
The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The example screens and verifies intestinal flora markers capable of being used for vitiligo diagnosis, an experimental flow is shown in figure 2, and the specific operation is as follows:
(1) And (5) collecting a sample.
Faecal samples of 79 vitiligo patients and 78 healthy persons (control group) were collected:
Vitiligo group samples were from 79 groups of households, and inclusion criteria were: ① Age distribution 3-65 years old; ② Patients clinically diagnosed with vitiligo; ③ The vital signs are stable.
The exclusion criteria for vitiligo groups were: ① General or local treatment of vitiligo is received 1 month ago; ② Systemic antibiotics, systemic hormones, cytokines and immunosuppressants were taken in the last three months; ③ A combination with other autoimmune diseases, gastrointestinal diseases, liver diseases, mental and psychological related diseases or other skin diseases; ④ Serious, life threatening diseases such as heart disease, kidney disease, endocrine system disease, cancer or immunodeficiency disease; ⑤ Pregnant, women of child-bearing age scheduled to become pregnant or lactating during the study; ⑥ Researchers consider patients unsuitable for inclusion in the study.
The control group samples are from 78 families, and the inclusion standard is ① years old and 3-65 years old; ② No patients with diabetes or other metabolic diseases; ③ No depression or other neurological disease; ④ No suffering from irritable bowel syndrome or gastrointestinal disease; ⑤ No other immune system diseases or no immunodeficiency state; ⑥ No antibiotics (e.g., neomycin, rifaximin) or probiotic prebiotics, etc. were taken prior to and during the study.
The control group was excluded from the standard and vitiligo group.
The above data were derived from stool samples collected by the martial arts hospitals.
(2) DNA extraction, library construction and sequencing.
The sample genomic DNA of the microorganism was extracted by CTAB (cetyltrimethylammonium bromide) method, followed by PCR amplification. The amplification primers were used for library construction using a TruSeq@DNA PCR-FREE SAMPLE Preparation Kit library Kit and then sequenced on-machine using Illumina Miseq PE.
(3) LEfSe analysis of screening markers.
Filtering, denoising and splicing the machine-down data to obtain high-quality sequencing data, and clustering by setting the Identity standard as 100% to divide ASV characteristic sequences. The feature sequence was annotated using the plugin feature-CLASSIFIER CLASSIFY-sklearn of qiime to generate taxonomic abundance data. Randomly selected 80% abundance data was analyzed using LEfSe software, default set the screening value for LDA Score to 3.5, and the results are shown in figure 2. Screening 3 microorganisms with significantly increased relative abundance in vitiligo group, namely Sagnac fungus genus Sutterella, maosporium Lachnospira and Paramycolatopsis genus Parabacteroides; and 3 microorganisms whose relative abundance is significantly reduced in the vitiligo group, namely, pediococcus Subdoligranulum, bifidobacterium and Blauthia. The box-type scatter plots for the six genera are shown in FIG. 3. These six genera were first found to be associated with vitiligo.
(4) And (5) establishing a predictive scoring system.
The remaining 20% abundance data was subjected to binary logistic regression and then subject work curve test (ROC curve) analysis to yield a cutoff value (optimal cut-off). The sample information table in steps (3) and (4) is shown in table 1.
Table 1 sample information table
The method comprises the steps of finishing specificity and sensitivity calculation and ROC curve drawing by utilizing IBM SPSS STATISTICS (v 27) statistical software, calculating a threshold value of an actual measurement value in the software, and then calculating the true positive example number (TP), the false positive example number (FP), the true negative example number (TN) and the false negative example number (FN) corresponding to the threshold value, wherein the specificity (true negative rate) =TN/(TN+FP), the sensitivity (true positive rate) =TP/(TP+FN), and constructing the ROC curve through 1-specificity and sensitivity, wherein the integral of the ROC curve is AUC. In order to calculate the specificity and sensitivity of a certain index, a about sign coefficient (about sign index=sensitivity+specificity-1) is calculated first, and the specificity and sensitivity corresponding to the maximum value of the about sign coefficient is the specificity and sensitivity of the certain index.
Subject work curve testing (ROC curve) analysis was performed with relative abundance values of single or multiple microbial markers, resulting in a cutoff value (optimal cut-off). From the analysis results (fig. 5 and table 2), it is known that the prediction ability of the genus Blautia (Blautia) on vitiligo is far higher than that of other genus, but the prediction effect of the six-bacterium combination is the highest; when six bacteria are combined, the AUC is 1.000, the optimal cut-off value is 0.500, the sensitivity is 100.0%, and the specificity is 100.0%.
TABLE 2ROC diagnostic Curve results
In summary, the invention discovers the relevance of procaryotes Sagnaporthe Sutterella, the chaetomium Lachnospira, the Paramycolatopsis Parabacteroides, the Pediococcus Subdoligranulum, the Bifidobacterium and the Blauthia of the Blauthia for the first time, and the 6 bacteria are used as detection markers for the diagnosis of the vitiligo, so that the accuracy is high and the diagnosis mode is noninvasive; therefore, the invention provides a new thought and approach for diagnosis and treatment of vitiligo.
It should be noted that the above-mentioned embodiments are only some embodiments of the present invention, but not all embodiments, and are only used for illustrating the technical scheme of the present invention, not limiting; all other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Claims (10)
1. The use of a microorganism of the intestinal flora as a marker for the detection of vitiligo, characterized in that the marker for the detection of vitiligo comprises Blautia of the genus Blautia.
2. The use according to claim 1, wherein the marker for detection of vitiligo consists of the genus salmeterol Sutterella, the genus Lachnospira, the genus bacteroides Parabacteroides, the genus micrococcus Subdoligranulum, the genus Bifidobacterium and the genus Blautia.
3. The use according to claim 2, wherein the abundance of said sarcandra Sutterella, lachnospira, paracoccidiopsis Parabacteroides is increased and the abundance of said rare micrococcus Subdoligranulum, bifidobacterium bifidobacteria, blautia is decreased in a vitiligo patient population compared to a healthy population.
4. The application of a vitiligo detection marker in preparing a product for diagnosing vitiligo is characterized in that the vitiligo marker comprises Blautia of Blautia.
5. The use according to claim 3, characterized in that the marker for detection of vitiligo consists of the genus satchel Sutterella, the genus Lachnospira, the genus bacteroides Parabacteroides, the genus micrococcus Subdoligranulum, the genus Bifidobacterium and the genus Blautia.
6. The use according to claim 4, wherein the product is a detection kit comprising reagents for detecting the abundance of vitiligo detection markers in a fecal sample of a subject.
7. The use according to claim 4, wherein the product is a diagnostic system having as a detection variable the abundance of vitiligo detection markers in a fecal sample of a subject.
8. The application of a vitiligo detection marker in preparing a medicament or a functional food for relieving vitiligo is characterized in that the vitiligo marker comprises Blautia of Bluet.
9. The use according to claim 8, wherein the marker for detection of vitiligo consists of the genus salmeterol Sutterella, the genus Lachnospira, the genus bacteroides Parabacteroides, the genus micrococcus Subdoligranulum, the genus Bifidobacterium and the genus Blautia.
10. A method for screening a medicament or functional food for treating or alleviating symptoms of vitiligo, characterized by screening a substance which causes the abundance of salmeterol genus Sutterella, chaetobacter Lachnospira, paracoccidiobacter Parabacteroides to decrease and/or causes the abundance of micrococcus rarefaciens Subdoligranulum, bifidobacterium bifidobactrium, blautia to increase in the intestinal tract, as a medicament or functional food for treating or alleviating the symptoms of vitiligo.
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