阅读说明：本技术 一种肌少症的肠道菌群标志物及其应用 (Intestinal flora marker for sarcopenia and application thereof ) 是由 康琳 于 2020-04-22 设计创作，主要内容包括：本发明公开了一种肌少症的肠道菌群标志物及其应用,本发明通过提取样本中的肠道菌群的DNA基因组并测序从而获得肠道菌群的种类和丰度特征,并基于肠道菌群的丰度特征进行肌少症的诊断。(The invention discloses a sarcopenia intestinal flora marker and application thereof.)

1. An intestinal flora marker for sarcopenia, which is characterized in that the intestinal flora marker is Anaerofilum.

2. Use of an intestinal microbiota marker according to claim 1 for the preparation of a product for the diagnosis of sarcopenia, wherein the product comprises a reagent for detecting the intestinal microbiota marker.

3. The use of claim 2, wherein the agent is a primer, probe, antisense oligonucleotide, aptamer or antibody specific for the gut flora marker.

4. The use of claim 3, wherein the primer is a primer that specifically amplifies 16SrRNA of the marker of gut flora.

5. A system for predicting sarcopenia using the gut flora marker of claim 1, comprising:

a nucleic acid sample separation unit for separating a nucleic acid sample of the intestinal flora from a detection object;

the sequencing unit is used for sequencing the separated intestinal flora nucleic acid sample to obtain a sequencing result;

the data processing unit is used for detecting the relative abundance of the microbial markers in the intestinal flora according to the sequencing result, and analyzing the obtained relative abundance value to obtain the critical value of the microbial markers;

and the result judging unit is used for comparing the critical value of the microbial marker obtained by the data processing unit with the set diagnostic value.

6. A product for diagnosing sarcopenia comprising a reagent for detecting the abundance of the gut flora marker of claim 1.

7. The product according to claim 6, wherein the reagent comprises a primer, probe, antisense oligonucleotide, aptamer or antibody specific for detecting the gut flora marker, preferably the specific primer is a primer capable of detecting the gut flora marker 16 SrRNA.

8. The product according to claim 6 or 7, further comprising a reagent for extracting genomic DNA, proteins and components of the microorganism.

9. Use of the gut flora marker of claim 1 for constructing a computational model for predicting sarcopenia.

10. Use of the gut flora marker of claim 1 for the preparation of a pharmaceutical composition for the treatment of sarcopenia.

Technical Field

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

Background

Sarcopenia (Sarcopenia), abbreviated Sarcopenia, is a degenerative syndrome caused by loss of skeletal muscle mass, reduction of muscle strength and function in the aging process, Sarcopenia causes a decrease in the activity ability, protein malnutrition, a decrease in the resting metabolic rate and thus causes emotional disturbances, furthermore, a decrease in the contractile ability of skeletal muscles causes a decrease in the load borne by bones, leaves bones in a relatively disused state for a long time, and is prone to induce osteoporosis (Buckwalter osteoporosis. osteoarrhiatus-Cartilage Use, Disuse, and Abuse-Experimental studies. JRheumatology. 1995; 22: 13-15.). Rong Hai et al have a Sarcopenia epidemiological investigation study in the area of Western China that in the age group of 60-70 years, the male prevalence rate is 8.2% and the female threat is 6.7%, in the group of more than 80 years, the healthy age group has an adverse effect similar to Borception of obesity L.437, and the rest of senile people who suffer from obesity, obesity.

The current research shows that factors such as skeletal muscle disuse, hormone level change, inflammatory response, insulin resistance and nutritional deficiency, and some chronic consumptive diseases are all related to the occurrence of Sarcopenia (Zembron-L acny A, Dziubek W et al, Sarcopenia: moniting, molecular mechanisms, and physical intervention. physical Res. 2014; 63(6):683 691.) has research showing that intestinal flora plays an important role in the occurrence and development of Sarcopenia, but the current research has less.

Disclosure of Invention

An object of the present invention is to provide a marker for intestinal flora for diagnosing sarcopenia.

The second purpose of the invention is to provide a product for diagnosing sarcopenia.

It is a further object of the present invention to provide a system for predicting sarcopenia.

The fourth purpose of the invention is to provide a pharmaceutical composition for treating sarcopenia.

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

the first aspect of the invention provides an intestinal flora marker for sarcopenia, and the intestinal flora marker is Anaerofilum.

In a second aspect, the invention provides the use of a marker of gut flora according to the first aspect of the invention for the manufacture of a product for diagnosing sarcopenia, said product comprising a reagent for detecting a marker of gut flora.

Further, the agent is a primer, probe, antisense oligonucleotide, aptamer or antibody specific for the gut flora marker.

Further, the primer is a primer for specifically amplifying the 16SrRNA of the intestinal flora marker.

A third aspect of the present invention provides a system for predicting sarcopenia using the gut flora markers of the first aspect of the present invention, comprising:

a nucleic acid sample separation unit for separating a nucleic acid sample of the intestinal flora from a detection object;

the sequencing unit is used for sequencing the separated intestinal flora nucleic acid sample to obtain a sequencing result;

the data processing unit is used for detecting the relative abundance of the microbial markers in the intestinal flora according to the sequencing result, and analyzing the obtained relative abundance value to obtain the critical value of the microbial markers;

and the result judging unit is used for comparing the critical value of the microbial marker obtained by the data processing unit with the set diagnostic value.

Using this system, the relative abundance of the biomarkers of the invention in the gut flora can be determined. Therefore, the probability of determining that the subject individual is a sarcopenia individual or a healthy individual can be improved by comparing the obtained relative abundance value with a predetermined critical value.

In a fourth aspect, the invention provides a product for diagnosing sarcopenia, the product comprising a reagent for detecting the abundance of an gut flora marker according to the first aspect of the invention.

Further, the reagent comprises a primer, a probe, an antisense oligonucleotide, an aptamer or an antibody for detecting the specificity of the intestinal flora marker.

Further, the specific primer is a primer capable of detecting the intestinal flora marker 16 SrRNA.

Furthermore, the product also comprises reagents for extracting microbial genome DNA, microbial protein and thallus components.

In a fifth aspect, the present invention provides a use of the marker of intestinal flora according to the first aspect of the present invention in constructing a computational model for predicting sarcopenia.

A sixth aspect of the invention provides the use of an intestinal flora marker according to the first aspect of the invention for the preparation of a pharmaceutical composition for the treatment of sarcopenia.

Further, the pharmaceutical composition comprises an agent that decreases the abundance of the gut flora marker.

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

The invention has the advantages and beneficial effects that:

the invention discovers that Anaerrofil is related to sarcopenia for the first time, the abundance of Anaerrofil is obviously different between sarcopenia patients and healthy people, and ROC curve analysis has higher specificity and sensitivity when being used as a detection variable, so that Anaerrofil can be used as a detection marker for diagnosing the sarcopenia patients. Anaerofilmum is used as a detection marker, so that the detection is completely noninvasive and high in accuracy.

Drawings

FIG. 1 is a graph of Rank-Absundance;

FIG. 2 is a graph of Anaerofilum differences;

fig. 3 is a ROC graph with analofilmum as the detection 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 diseases is found, the intestinal flora and the disease information are integrated, and the sarcopenia patients are predicted to the greatest extent. According to the invention, the significant difference of Anaerofilm in sarcopenia patients and healthy people is found for the first time through 16S rRNA sequencing, which indicates that Anaerofilm can be used as a prediction factor of sarcopenia.

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 "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 relate to amplification using the polymerase chain reaction ("PCR"), many other methods known in the art for amplifying nucleic acids (e.g., isothermal methods, rolling circle methods, etc.) are also encompassed by the present invention. 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 "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 "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.

As used herein, the term "stringency" is used to refer to conditions of temperature, ionic strength, and presence of other compounds under which nucleic acid hybridization will occur only between nucleic acids having sufficiently long fragments with a high frequency of complementary base sequences.exemplary hybridization conditions are as follows.high stringency generally refers to conditions that allow only those nucleic acids that form stable hybridization products to hybridize at 65 ℃ and in 0.018M NaCl.for example, high stringency conditions can be provided by hybridizing in 50% formamide, 5 × Denhardt's solution, 5 × SSC (sodium citrate saline), 0.2% SDS (sodium dodecyl sulfate) and at 42 ℃, followed by washing in 0.1% SDS and at 65 ℃.moderate stringency refers to conditions equivalent to hybridizing in 50% formamide, 5 × Denhardt's solution, 5 ×, 0.2% SDS and after 42 ℃ SDS, 4832% SDS and 0.38710% SDS, after hybridizing in 50% formamide, 5 Denhardt solution, 5% SDS and 0.1% SDS at × ℃ and after hybridizing in 0.48 ℃ SDS, the same conditions are equivalent to 50% formamide, 5% SDS, 3.3.48% SDS and 5% SDS, 3.863% SDS.

As used herein, a "primer" for amplification is an oligonucleotide that specifically anneals to a target nucleotide sequence or a labeled nucleotide sequence. The 3' nucleotide of the primer should be identical to the target sequence or tag sequence at the corresponding nucleotide position for optimal primer extension by the polymerase.

In the present invention, the term "antibody" is used in the broadest sense and specifically covers, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single chain antibodies, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

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 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.