阅读说明：本技术 诊断肌少症的肠道菌群及其应用 (Intestinal flora for diagnosing sarcopenia and application thereof ) 是由 康琳 于 2020-04-22 设计创作，主要内容包括：本发明公开了诊断肌少症的肠道菌群及其应用,所述肠道菌群为Eisenbergiella。本发明通过16S核糖体核糖核酸测序首次发现了Eisenbergiella在肌少症中呈现显著性上调,进一步发现了使用Eisenbergiella作为检测变量区分肌少症和健康群体具有较高的特异性和敏感性,基于此,可以将Eisenbergiella应用于肌少症的无创诊断。(The invention discloses an intestinal flora for diagnosing sarcopenia and application thereof, wherein the intestinal flora is Eisenbergiella. According to the invention, the fact that Eisenbergiella is remarkably up-regulated in sarcopenia is firstly discovered through 16S ribosomal ribonucleic acid sequencing, and the fact that Eisenbergiella is used as a detection variable to distinguish sarcopenia from healthy groups is further discovered to have higher specificity and sensitivity, so that Eisenbergiella can be applied to noninvasive diagnosis of sarcopenia.)

1. Use of one or more reagents for detecting the abundance of gut flora comprising at least the gut flora Eisenbergiella in the manufacture of a product for predicting sarcopenia.

2. The use according to claim 1, wherein the agent comprises one or more oligonucleotides capable of specifically hybridizing to a target nucleotide sequence from Eisenbergiella.

3. The use according to claim 1, wherein the product further comprises a reagent for isolating nucleic acids from the sample.

4. Use according to claim 2, wherein the oligonucleotide is detectably labelled.

5. The use according to claim 2, wherein the target nucleotide sequence of Eisenbergiella is a fragment of the 16S ribosomal ribonucleotide gene.

6. A kit for predicting sarcopenia is characterized in that the kit comprises a reagent for detecting the abundance of the intestinal flora Eisenbergiella.

7. The kit of claim 6, wherein the reagents comprise one or more oligonucleotides capable of specifically hybridizing to a target nucleotide sequence from Eisenbergiella.

8. The kit of claim 7, wherein the oligonucleotide comprises a probe that specifically recognizes the target nucleotide sequence of Eisenbergiella or a primer that specifically amplifies the target nucleotide sequence of Eisenbergiella.

9. The kit of claim 8, wherein the amplification is PCR or RT-PCR, preferably wherein the amplification utilizes detectably labeled primers.

10. A composition and use thereof in the manufacture of a medicament for the treatment of sarcopenia, wherein the composition comprises an agent that reduces the abundance of Eisenbergiella.

Technical Field

The invention belongs to the technical field of biology, and relates to intestinal flora for diagnosing sarcopenia and application thereof.

Background

Muscular attenuation syndrome (Sarcopenia), abbreviated Sarcopenia, originally proposed by Evans WJ and Rosenberg IR in 1991, and currently gaining more and more importance 2010, European Sarcopenia working group proposed the definition that Sarcopenia is a progressive loss of muscle mass and strength, which may lead to limb disability, decreased quality of life and even death (Cruz-Jentoft AJ, Baeyens JP, Bauer JM, et al. Sarcopenia: European symphysis on definition and diagnosis: report of the European bone fibrosis on diagnosis and diagnosis: report of the European bone fibrosis in muscle tissue, 2010,39(4): 412. 423. Sarcopenia. diagnosis about Sarcopenia, many studies can be made by measuring grip strength, number of steps, etc., but no more than the Sarcopenia, like, found by research on the physiological activities of human beings, such as the physiological activities of diabetes mellitus, physiological activities of physiological diseases [ 12. the research of physiological activities of human beings, research [ 12. the research of physiological activities of obesity, research [ 12. the research of physiological activities of human beings, research [ 12. the physiological activities of diabetes, research of human beings, research [ 12. the same, research of the diseases of the physiological activities of the diseases of human beings), biological, research [ 12. the same, research of the human beings), the diseases of human beings, research [ 1. the diseases of the research [ 1. the same, the diseases of human beings, the diseases of the human beings No. 1. the diseases of the human beings No. 1. the diseases of the human beings, the diseases of.

The number of microorganisms known at present far exceeds the number of human cells, and most microorganisms in humans reside in the intestinal tract, which play a role in intestinal barrier, immune and endocrine function by mediating various host reactions (binders L B, Delzenne NM. muscle walking: the gut microbiota as a new intestinal target.

Disclosure of Invention

The invention aims to provide intestinal flora related to the occurrence and development of sarcopenia and application thereof in diagnosing and treating sarcopenia.

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

the invention provides the use of one or more reagents for detecting the abundance of gut flora including at least the gut flora Eisenbergiella in the manufacture of a product for predicting sarcopenia.

Further, the reagents include one or more oligonucleotides capable of specifically hybridizing to a target nucleotide sequence from Eisenbergiella.

Further, the product may further comprise a reagent for isolating nucleic acid from the sample.

Further, the oligonucleotide is detectably labeled.

Further, the target nucleotide sequence of Eisenbergiella is a fragment of the 16S ribosomal ribonucleic acid gene.

The invention provides a kit for predicting sarcopenia, which comprises a reagent for detecting the abundance of intestinal flora Eisenbergiella.

Further, the reagents include one or more oligonucleotides capable of specifically hybridizing to a target nucleotide sequence from Eisenbergiella.

Further, the oligonucleotide includes a probe that specifically recognizes the target nucleotide sequence of Eisenbergiella or a primer that specifically amplifies the target nucleotide sequence of Eisenbergiella.

Further, the amplification is PCR or RT-PCR, preferably, the amplification utilizes detectably labeled primers.

The invention provides a composition and application thereof in preparing a medicament for treating sarcopenia, wherein the composition comprises an agent for reducing the abundance of Eisenbergiella.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

Drawings

FIG. 1 is a graph of Rank-Absundance;

FIG. 2 is a graph of the differences of Eisenbergiella;

FIG. 3 is a ROC plot of Eisenbergiella as the sensed variable.

Detailed Description

In order to evaluate whether the composition of the intestinal symbiotic flora can be used as a prediction factor of sarcopenia, samples of sarcopenia patients and healthy people are collected, 16S rRNA sequencing is carried out, statistics of sequencing data is carried out by using bioinformatics, the intestinal flora related to sarcopenia is screened out, the intestinal flora and disease information are integrated, and the sarcopenia patients are predicted to the greatest extent. According to the invention, through 16S rRNA sequencing, the remarkable difference of the Eisenbergiella in sarcopenia patients and healthy people is found for the first time, and the Eisenbergiella can be used as a prediction factor of sarcopenia.

In an embodiment of the present invention, the present invention diagnoses sarcopenia by: detecting one or more nucleic acid fragments corresponding to a species that is diagnostic of sarcopenia in a nucleic acid sample from an individual. In a particular embodiment, nucleic acid fragments corresponding to Eisenbergiella 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.

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, sarcopenia is diagnosed in a subject based on analyzing a microbiota 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 stool.

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 Eisenbergiella. An example of a clinical specimen is stool. 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.