Kit for evaluating healthy young and healthy senescence of people and model construction method thereof

Abstract

The invention provides a kit for evaluating healthy young and healthy senescence of people, the kit contains a reagent for detecting intestinal bacteria Gordonibacter or C. scinders, the sequence of a forward primer for detecting DNA of the Gordonibacter is shown as SEQ ID NO.1, the sequence of a reverse primer for detecting DNA of the Gordonibacter is shown as SEQ ID NO.2, the sequence of a forward primer for detecting DNA of the C. scinders is shown as SEQ ID NO.3, and the sequence of a reverse primer for detecting DNA of the C. scinders is shown as SEQ ID NO.4. The invention further provides a kit for evaluating healthy young and healthy senescence of people. If the absolute content of the Gordonibacter or the absolute content of the C. scinders or the absolute content of the Gordonibacter and the absolute content of the C. scinders in the excrement are all larger than 1000 copy numbers per milligram of the excrement, it is shown that the detected object is in a healthy young state, and otherwise, it is shown that the detected object is in a healthy senescence state. The invention further provides a construction method of the model for predicting healthy young and healthy senescence of the human body. By utilizing the kit provided by the invention, the healthy young and healthy aging states of a subject can be quantitatively evaluated according to the absolute abundance of two bacteria (Gordonibacter and C. scinders) in the excrement of the subject.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a kit, in particular to a kit for evaluating healthy youth and healthy aging of a person and a model construction method thereof. Background Art

[0002] As people age, their physiological functions gradually decline, a process often accompanied by a variety of physiological changes and the occurrence of chronic diseases. During the physiological aging process, the human body is often accompanied by a decline in organ function. Different from pathological organ dysfunction (often accompanied by the occurrence of disease), this physiological organ dysfunction that increases with age is also called physiological / healthy aging. However, in clinical practice, it is difficult to accurately and non-invasively identify healthy youth or healthy aging. In recent years, more and more studies have found that intestinal microbial communities play an important role in promoting human aging.

[0003] Our study found that the abundance of Gordonibacter and C.scindens in the intestine increased significantly during the aging process. This change is related to human aging and aging-related organ dysfunction, and can predict the healthy aging state of the human body.

[0004] Therefore, accurate assessment of the abundance of these microorganisms in the intestine provides new ideas and non-invasive and rapid assessment strategies for the prediction, intervention and treatment of healthy aging and aging-related organ dysfunction.

[0005] Gordonibacter: Gordonibacterium, Gram-positive, short rod / coccus-shaped anaerobic bacteria.

[0006] C.scindens: C.scindens, a bacterium of the genus Clostridium, a Gram-positive clostridial anaerobic bacterium. Summary of the invention

[0007] In response to the above-mentioned technical problems in the prior art, the present invention provides a kit for evaluating human health youth and healthy aging, as distinguished from youth / aging under disease conditions, and a model construction method thereof. The kit for evaluating human health youth and healthy aging and a model construction method thereof are intended to solve the technical problem that there is no rapid and non-invasive method for evaluating human health youth and healthy aging in the prior art.

[0008] The present invention provides a kit for evaluating healthy youth and healthy aging of a person, wherein the kit contains a reagent for detecting intestinal bacteria Gordonibacter or C.scindens, and the reagent contains a forward primer and a reverse primer for detecting DNA of Gordonibacter or C.scindens.

[0009] The sequence of the forward primer for detecting Gordonibacter DNA is:

[0010] 5'-AGCGGGACAGAGATAGGATAGT-3' (SEQ ID NO. 1);

[0011] The sequence of the reverse primer for detecting Gordonibacter DNA is:

[0012] 5'-AGAGCTTCGGGTTATCACTAAGT-3' (SEQ ID NO. 2);

[0013] The sequence of the forward primer for detecting C.scindens DNA is:

[0014] 5'-GAACAGACGCAGGAAAGGA-3' (SEQ ID NO. 3);

[0015] The sequence of the reverse primer for detecting C.scindens DNA is:

[0016] 5'-TAGCCAAACGTCACCCAGAG-3' (SEQ ID NO. 4).

[0017] Furthermore, using feces as the test object, if the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces is greater than 1000 copies per milligram of feces, it indicates that the test subject is in a healthy aging state; otherwise, it indicates that the test subject is in a healthy young state.

[0018] The present invention also provides the use of intestinal bacteria Gordonibacter or C.scindens as a biomarker in the preparation of a kit for evaluating healthy youth and healthy aging of a human.

[0019] Furthermore, using feces as the test object, if the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces is greater than 1000 copies per milligram of feces, it indicates that the test subject is in a healthy aging state; otherwise, it indicates that the test subject is in a healthy young state.

[0020] The present invention also provides a model for predicting healthy youth and healthy aging of the human body, wherein the input variable of the model is the abundance of the detected intestinal bacteria Gordonibacter or C.scindens.

[0021] The present invention also provides a method for constructing a model for predicting healthy youth and healthy aging of the human body, comprising the following steps:

[0022] 1) Screening of differential bacteria under healthy aging and aging-related organ function decline:

[0023] Fecal samples were collected from healthy young people and healthy aging people, and metagenomic sequencing was used to detect the intestinal flora of healthy young people and healthy aging people, to obtain differential bacteria between healthy young people and healthy aging people, and the bacteria Gordonibacter and C.scindens were found as potential biological markers to distinguish the two groups of people;

[0024] 2) Primer design and validation for specific Gordonibacter and C.scindens:

[0025] To obtain specific primers for Gordonibacter and C. scindens , representative genomes of the corresponding target bacteria were downloaded from NCBI, and the core gene sequences of the representative genomes of the target bacteria were identified and conserved sequences were selected using the bioinformatics software “Roary”, “Prank”, and “consambig” in turn;

[0026] The core gene sequences of the target bacteria were compared using the "BLAST+" software and the NCBI bacterial reference genome sequence database, and the core gene sequences that were conserved and single-copy in the target bacteria were used as candidate genes for subsequent primer design;

[0027] For representative gene sequences of conservative and single-copy target bacteria, “Primer3” was used for primer design. After the design was completed, the algorithms “BLAST+” and “mFold” were used for quality control and preliminary verification of the primers.

[0028] Then, the available primer pairs selected by the above algorithm are sorted by the primer penalty provided by Primer3, and the primers with the lowest penalty are selected for subsequent experimental verification;

[0029] The sequence of the forward primer for detecting Gordonibacter DNA is:

[0030] 5'-AGCGGGACAGAGATAGGATAGT-3' (SEQ ID NO. 1);

[0031] The sequence of the reverse primer for detecting Gordonibacter DNA is:

[0032] 5'-AGAGCTTCGGGTTATCACTAAGT-3' (SEQ ID NO. 2);

[0033] The sequence of the forward primer for detecting C.scindens DNA is:

[0034] 5'-GAACAGACGCAGGAAAGGA-3' (SEQ ID NO. 3);

[0035] The sequence of the reverse primer for detecting C.scindens DNA is:

[0036] 5'-TAGCCAAACGTCACCCAGAG-3' (SEQ ID NO. 4).

[0037] 3) Specific detection of target primers:

[0038] The whole genome DNA of the bacterial flora of the positive samples and the negative control samples was extracted, and the selected primers were used to perform polymerase chain reaction combined with gel electrophoresis analysis and real-time quantitative polymerase chain reaction combined with melting curve analysis on the negative samples and the positive samples to detect the specificity of the primers;

[0039] 4) Specificity Gordonibacter and C.scindens sensitivity:

[0040] The known concentrations of standard DNA amplified fragments were diluted in equal proportions and the linear range of detection for two specific bacteria was found to be: 10 2 Up to 10 8 The minimum detection limit of the reaction system is 100 copies per microliter of sample volume, and the maximum detection limit is 10 8 Number of copies per microliter of sample;

[0041] 5) Detect Gordonibacter and C.scindens in the intestinal feces of actual subjects and evaluate the indicator boundaries between healthy youth and healthy aging;

[0042] The standard curve method real-time quantitative PCR was performed on the genomic DNA of fecal flora of the two groups of people using primers, and the ratio of DNA copy number to milligrams of feces was used to represent the absolute content of bacteria per milligram of feces;

[0043] The results showed that the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces of the healthy aging population was greater than 1000 copies per milligram of feces, suggesting that greater than 1000 copies per milligram of feces can be used as a demarcation value for defining the changes between healthy youth and healthy aging in the population.

[0044] The present invention also provides a reagent for detecting intestinal bacteria Gordonibacter or C.scindens for non-diagnostic and non-therapeutic purposes, wherein the reagent contains a forward primer and a reverse primer for detecting DNA of Gordonibacter or C.scindens.

[0045] The sequence of the forward primer for detecting Gordonibacter DNA is:

[0046] 5'-AGCGGGACAGAGATAGGATAGT-3' (SEQ ID NO. 1);

[0047] The sequence of the reverse primer for detecting Gordonibacter DNA is:

[0048] 5'-AGAGCTTCGGGTTATCACTAAGT-3' (SEQ ID NO. 2);

[0049] The sequence of the forward primer for detecting C.scindens DNA is:

[0050] 5'-GAACAGACGCAGGAAAGGA-3' (SEQ ID NO. 3);

[0051] The sequence of the reverse primer for detecting C.scindens DNA is:

[0052] 5'-TAGCCAAACGTCACCCAGAG-3' (SEQ ID NO. 4).

[0053] The present invention also provides a combination of microbial markers for diagnosing or predicting healthy youth and healthy aging of the human body, which are intestinal bacteria Gordonibacter and / or C. scindens.

[0054] The present invention also provides a system for predicting healthy youth and healthy aging of a human body, comprising the following modules:

[0055] A data input module, used to input the abundance data of the microbial marker combination obtained in the biological sample of the subject, wherein the microbial marker combination is intestinal bacteria Gordonibacter and / or C.scindens;

[0056] The prediction module is connected to the data input module, sets the boundaries of healthy youth and healthy aging indicators of Gordonibacter and C.scindens, and predicts whether the subject is healthy youth or healthy aging based on the abundance data of the microbial marker combination of the subject obtained from the data input module.

[0057] Specifically, for healthy young people, it is defined as: age ≤60 years old and no obvious liver, kidney and lung function decline is found in any two tests within one month. Specifically, in any two tests within one month: liver function alanine aminotransferase test index is <35U / L, aspartate aminotransferase test index is <35U / L, and renal function test glomerular filtration rate is ≥90 / mL / min / m 2 , the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1 / FVC) in lung function test is ≥85%, and the percentage of forced vital capacity FEV1 to predicted value (%FEV1) is ≥85%;

[0058] For healthy aging people, it is defined as: those who are over 60 years old or have obvious liver, kidney and lung function decline in any two tests within one month. Specifically, in any two tests within one month: in liver function test, 35U / L≤alanine aminotransferase test index ≤50U / L, 35U / L≤aspartate aminotransferase test index ≤50U / L, in kidney function test, glomerular filtration rate <90 / mL / min / m 2 , the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1 / FVC) in lung function test is <85%, and the percentage of forced vital capacity FEV1 to the predicted value (%FEV1) is <85%).

[0059] The predicted value refers to the predicted forced expiratory volume in the first second of a patient, which is automatically calculated after the patient's age, height, gender and weight parameters are input into the machine for detecting lung function.

[0060] The present invention uses a kit for evaluating the healthy youth and healthy aging of the human body by detecting the abundance of specific bacteria in feces through a specific primer combination. The present invention can accurately determine the abundance of Gordonibacter and C.scindens in the intestine, and can evaluate healthy youth and healthy aging based on this information. The method is rapid, non-invasive, highly accurate, and highly sensitive, and can be used for the evaluation of healthy youth and healthy aging in clinical and research fields.

[0061] Compared with the prior art, the technical effect of the present invention is positive and obvious. The kit provided by the present invention can be used to quantitatively evaluate the state of healthy youth and healthy aging of the subject based on the absolute abundance of two bacteria (Gordonibacter and C.scindens) in the feces of the subject, providing a new idea and non-invasive detection method for the detection of healthy youth and healthy aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 .Differential bacteria in healthy aging and aging-related organ function decline based on the LEfSe algorithm.

[0063] Figure 2 .Polymerase chain reaction and gel electrophoresis results, where: Lane 1 is the quality control electrophoresis of the Gordonibacter negative stool sample system (amplification using bacterial universal primers 338-F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806-R: 5'-GGACTACHVGGGTWTCTAAT-3'); Lane 2 is the PCR and electrophoresis analysis of Gordonibacter positive samples (Gordonibacter low penalty primers); Lane 3 is the Gordonibacter Lane 1 is the PCR and electrophoresis analysis of C. scindens negative stool samples (using Gordonibacter low penalty primers); Lane 4 is the quality control electrophoresis of the C. scindens negative stool sample system (amplification using bacterial universal primers 338-F and 806-R); Lane 5 is the PCR and electrophoresis analysis of C. scindens positive stool samples (using C. scindens low penalty primers); Lane 6 is the PCR and electrophoresis analysis of C. scindens negative stool samples (using C. scindens low penalty primers).

[0064] Figure 3 Melting curves of the specific primers for Gordonibacter and C.scindens.

[0065] Figure 4 .The standard curve of this kit for detecting different concentrations of standard products of two bacteria.

[0066] Figure 5 .The kit was used to detect Gordonibacter and C.scindens in the intestinal feces of actual subjects, and the detection cut-off values ​​of healthy youth and healthy aging were evaluated. DETAILED DESCRIPTION

[0067] Example 1 Screening of differential bacteria under healthy aging and aging-related organ function decline:

[0068] To establish the association between age and healthy aging, we collected stool samples from 66 healthy people, including 33 healthy young people and 33 healthy aging people.

[0069] The definition of healthy people in this implementation is: 1) non-obese / body mass index (BMI) less than 28kg / m 2 ; 2) According to the questionnaire report and community medical history, the subjects do not have and have never had a clinical diagnosis of cancer, autoimmune disease, musculoskeletal disease, diabetes, endocrine disease, cardiovascular disease or other chronic diseases; 3) Have not taken anti-inflammatory drugs in the previous two weeks and have not taken antibiotics within two months; 4) The patient's current physical condition is stable, with no obvious illness, frailty or cognitive impairment.

[0070] To characterize healthy aging in the human body, we use chronological aging (age) combined with relevant organ function decline (a state of aging with persistent organ function impairment) as indicators of healthy aging in the human body.

[0071] Healthy young people are defined as those aged 60 years or younger and no obvious liver, kidney or lung function decline is found in any two tests within one month. Specifically, in any two tests within one month: the liver function test index of alanine aminotransferase is <35U / L, the test index of aspartate aminotransferase is <35U / L, and the glomerular filtration rate in the renal function test is ≥90mL / min / m 2 , the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1 / FVC) in lung function test is ≥85%, and the percentage of forced vital capacity FEV1 to predicted value (%FEV1) is ≥85%;

[0072] For healthy aging people, it is defined as: those who are over 60 years old or have obvious liver, kidney and lung function decline in any two tests within one month. Specifically, in any two tests within one month: in liver function test, 35U / L≤alanine aminotransferase test index ≤50U / L, 35U / L≤aspartate aminotransferase test index ≤50U / L, in kidney function test, glomerular filtration rate <90mL / min / m 2 , the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1 / FVC) in lung function test is <85%, and the percentage of forced vital capacity FEV1 to the predicted value (%FEV1) is <85%).

[0073] The predicted value refers to a patient's predicted forced expiratory volume in the first second value that is automatically calculated after the patient's age, height, gender, and weight parameters are input into a machine for detecting lung function (this is the prior art).

[0074] We used metagenomic sequencing to detect the intestinal flora of the two groups and annotated the metagenomic data using the HUMAnN3 algorithm. Furthermore, the linear discriminant analysis effect size algorithm was used to obtain the differential bacteria between the healthy young and healthy aging groups. Among them, bacteria with a linear discriminant analysis score (LDAScore) greater than or equal to 4 (Gordonibacter and C.scindens) were used as potential biomarkers to distinguish the two groups of people (such as Figure 1 Figure 2 shows the top 5 bacteria with the highest scores that were enriched in the elderly population.

[0075] Example 2 Primer design and verification of specific Gordonibacter and C. scindens

[0076] To obtain specific primers for Gordonibacter and C. scindens, we downloaded representative genomes of the corresponding target bacteria from NCBI (the representative genome of Gordonibacter was obtained with an ID of ASM334038 and the representative genome of C. scindens was obtained with an ID of ASM429512), and used the bioinformatics software "Roary", "Prank" and "consambig" to identify the core gene sequences of the representative genomes of the target bacteria and select conserved sequences (the parameters in the software used the default parameters).

[0077] The core gene sequences of the target bacteria were compared using the "BLAST+" software and the NCBI bacterial reference genome sequence database (default parameters). The core gene sequences were used as candidate genes for the conservative and single-copy gene sequences of the target bacteria for subsequent primer design (in this step, the number of single-copy gene sequences of Gordonibacter screened was 1618; the number of single-copy gene sequences of C. scindens was 1097).

[0078] For representative gene sequences of conservative and single-copy target bacteria, "Primer3" was used for primer design. After the design was completed, the algorithms "BLAST+" and "mFold" were used to perform quality control and preliminary verification of the primers. For Gordonibacter, 2 pairs of usable primers were screened; for C. scindens, 3 pairs of usable primers were screened (Table 1).

[0079] Table 1 Primer quality control items and the number of available primers after screening

[0080]

[0081] The available primer pairs screened by the algorithm were sorted by the primer penalty provided by Primer3, and the primers with the lowest penalty were selected (Table 2) for subsequent experimental verification.

[0082] Table 2 Minimum penalty primers for target bacteria

[0083]

[0084] Example 3 Specific detection of target primers:

[0085] The whole genomic DNA of the bacterial flora of positive samples (fecal samples known to contain Gordonibacter / C.scindens) and negative control samples (fecal samples known to not contain Gordonibacter / C.scindens) was extracted. The negative and positive samples were analyzed by polymerase chain reaction combined with gel electrophoresis using two selected low-penalty primers ( Figure 2 ) and real-time quantitative polymerase chain reaction combined with melting curve analysis ( Figure 3 ), to detect the specificity of the primers. The results showed that the two pairs of primers were specific to specific bacteria. The specific PCR amplification reaction conditions are shown in Table 3.

[0086] Specifically, the polymerase chain reaction combined with gel electrophoresis analysis results ( Figure 2 ) showed that the specific Gordonibacter primers had specific amplification for the positive samples (positive band in lane 2) and no non-specific amplification for the negative samples (no positive band in lane 3); the specific C.scindens primers had specific amplification for the positive samples (lane 5) and no non-specific amplification for the negative samples (lane 6); the quality control lanes (lanes 1 and 4) used bacterial universal primers 338-F and 806-R to perform quality control on the negative samples to exclude false negative results caused by sample preparation, and further exclude cross-reaction with other intestinal bacterial genomes (no non-specific amplification of other bacterial genomes in stool specimens without Gordonibacter / C.scindens).

[0087] Real-time quantitative polymerase chain reaction combined with melting curve analysis (results such as Figure 3 ) showed that the primers for specific Gordonibacter / C.scindens presented a single peak in the detection of specific bacteria in feces, indicating that both specific primers targeted specific sequences on specific bacteria and ensured the uniqueness of the amplified sequence, eliminating the false positive problem caused by multiple copies.

[0088] The results of polymerase chain reaction combined with gel electrophoresis analysis ( Figure 2) were detected using stool samples that did not contain the target bacteria, and there were no nonspecific bands, demonstrating that the primers were specific for Gordonibacter and C.scindens and did not cross-react with other intestinal bacteria.

[0089] Table 3. PCR and quantitative PCR reaction temperatures and conditions

[0090]

[0091]

[0092] Example 4 Sensitivity of specific Gordonibacter and C.scindens:

[0093] The standard DNA amplified fragments with known concentrations were diluted in equal proportions (dilution range: 10 9 ~1) detection, it was found that the detection linear range of the kit for two specific bacteria is: 10 2 Up to 10 8 The minimum detection limit of the reaction system is 100 copies per microliter of sample volume, and the maximum detection limit is 10 8 Copies per microliter of sample ( Figure 4 ).

[0094] Example 5 Gordonibacter and C.scindens in the intestinal feces of actual subjects were detected, and the boundaries between healthy youth and healthy aging indicators were evaluated.

[0095] Using the 66 healthy population samples in Implementation 1, the absolute quantitative cut-off values ​​of Gordonibacter and C.scindens in healthy young and healthy elderly populations were determined.

[0096] The standard curve method real-time quantitative PCR was performed on the genomic DNA of fecal flora of the two groups of people using primers (Table 3), and the ratio of DNA copy number to milligrams of feces was used to represent the absolute content of bacteria per milligram of feces.

[0097] The results showed that the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces of healthy aging people was greater than 1000 copies per milligram of feces ( Figure 5 ), suggesting that a copy number greater than 1000 per milligram of feces can be used as a cut-off value for defining healthy youth and healthy aging in the population.

[0098] Example 6 Accuracy of Gordonibacter and C.scindens in assessing healthy youth and healthy aging in independent populations.

[0099] We randomly recruited and collected stool from 65 independent healthy people. The intestinal flora genomic DNA was extracted from the collected stool, and the copy numbers of Gordonibacter and C.scindens in their stool were detected by real-time quantitative polymerase chain reaction using the standard curve method (Table 3). The healthy aging population was defined based on the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens (copy number per mg of feces) greater than 1000.

[0100] The test results of this implementation method are compared with the actual results (the healthy aging population is evaluated according to the standards in Specific Implementation 1) to calculate the specificity and sensitivity of the test. The specificity and sensitivity of this method for the detection of healthy aging population are shown in Table 4.

[0101] Table 4

[0102]

[0103]

[0104] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A kit for assessing healthy youth and healthy aging of a person, characterized in that: The kit contains a reagent for detecting intestinal bacteria Gordonibacter or C.scindens, and the reagent contains a forward primer and a reverse primer for detecting DNA of Gordonibacter or C.scindens. The sequence of the forward primer for detecting Gordonibacter DNA is: 5'-AGCGGGACAGAGATAGGATAGT-3'; The sequence of the reverse primer for detecting Gordonibacter DNA is: 5'-AGAGCTTCGGGTTATCACTAAGT-3'; The sequence of the forward primer for detecting C.scindens DNA is: 5′-GAACAGACGCAGGGAAAGGA-3′; The sequence of the reverse primer for detecting C.scindens DNA is: 5′-TAGCCAAACGTCACCCAGAG-3′.

2. A kit for assessing healthy youth and healthy aging of a person according to claim 1, characterized in that: When feces are used as test objects, if Gordonibacter or C.scindens or Gordonibacter+C. If the absolute content of scindens is greater than 1000 copies per mg of feces, it indicates that the subject is in a healthy aging state; otherwise, it indicates that the subject is in a healthy young state.

3. Use of intestinal bacteria Gordonibacter or C.scindens as biomarkers in the preparation of a kit for assessing healthy youth and healthy aging in humans.

4. The use according to claim 3, characterized in that: Using feces as the test object, if the absolute content of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces is greater than 1000 copies per milligram of feces, it shows that the test subject is in a healthy aging state. Otherwise, it means that the test subject is in a healthy young state.

5. A model for predicting healthy youth and healthy aging of the human body, characterized in that: The input variable of the model is the abundance of the detected intestinal bacteria Gordonibacter or C. scindens.

6. A method for constructing a model for predicting healthy youth and healthy aging of the human body, characterized in that: The steps include: 1) Screening of differential bacteria under healthy aging and aging-related organ function decline: Fecal samples were collected from healthy young people and healthy aging people, and metagenomic sequencing was used to detect the intestinal flora of healthy young people and healthy aging people, to obtain differential bacteria between healthy young people and healthy aging people, and the bacteria Gordonibacter and C.scindens were found as potential biological markers to distinguish the two groups of people; 2) Primer design and validation for specific Gordonibacter and C.scindens: To obtain specific primers for Gordonibacter and C. scindens , representative genomes of the corresponding target bacteria were downloaded from NCBI, and the core gene sequences of the representative genomes of the target bacteria were identified and conserved sequences were selected using the bioinformatics software "Roary", "Prank" and "consambig" in turn; Use "BLAST+" software and NCBI's bacterial reference genome sequence database to compare the core gene sequences of the target bacteria, and use the core gene sequences that are conservative and single-copy gene sequences of the target bacteria as candidate genes for subsequent primer design; For representative gene sequences of conservative and single-copy target bacteria, "Primer3" was used for primer design. After the design was completed, the algorithms "BLAST+" and "mFold" were used for quality control and preliminary verification of the primers. Then, the available primer pairs selected by the above algorithm are sorted by the primer penalty provided by Primer3, and the primers with the lowest penalty are selected for subsequent experimental verification; The sequence of the forward primer for detecting Gordonibacter DNA is: 5'-AGCGGGACAGAGATAGGATAGT-3'; The sequence of the reverse primer for detecting Gordonibacter DNA is: 5'-AGAGCTTCGGGTTATCACTAAGT-3'; The sequence of the forward primer for detecting C.scindens DNA is: 5′-GAACAGACGCAGGGAAAGGA-3′; The sequence of the reverse primer for detecting C.scindens DNA is: 5′-TAGCCAAACGTCACCCAGAG-3′. 3) Specific detection of target primers: The whole genome DNA of the bacterial flora of the positive samples and the negative control samples was extracted, and the selected primers were used to perform polymerase chain reaction combined with gel electrophoresis analysis and real-time quantitative polymerase chain reaction combined with melting curve analysis on the negative samples and the positive samples to detect the specificity of the primers; 4) Specificity Gordonibacter and C.scindens sensitivity: The known concentrations of standard DNA amplified fragments were diluted in equal proportions and the linear range of detection for two specific bacteria was found to be: 10 2 Up to 10 8 The minimum detection limit of the reaction system is 100 copies per microliter of sample volume, and the maximum detection limit is 10 8 Number of copies per microliter of sample; 5) Detect Gordonibacter and C.scindens in the intestinal feces of actual subjects and evaluate the indicator boundaries between healthy youth and healthy aging; The standard curve method real-time quantitative PCR was used to detect the genomic DNA of fecal flora in the two groups of people using primers, and the ratio of DNA copy number to milligrams of feces was used to represent the absolute content of bacteria per milligram of feces; The results showed that the absolute contents of Gordonibacter or C.scindens or Gordonibacter+C.scindens in the feces of the healthy aging population were all greater than 1000 copies per milligram of feces, suggesting that greater than 1000 copies per milligram of feces can be used as a demarcation value for defining the changes between healthy young people and healthy aging people.

7. A reagent for detecting intestinal bacteria Gordonibacter or C.scindens for non-diagnostic and therapeutic purposes, characterized in that: The reagent contains a forward primer and a reverse primer for detecting the DNA of Gordonibacter or C.scindens. The sequence of the forward primer for detecting Gordonibacter DNA is: 5'-AGCGGGACAGAGATAGGATAGT-3'; The sequence of the reverse primer for detecting Gordonibacter DNA is: 5'-AGAGCTTCGGGTTATCACTAAGT-3'; The sequence of the forward primer for detecting C.scindens DNA is: 5′-GAACAGACGCAGGGAAAGGA-3′; The sequence of the reverse primer for detecting C.scindens DNA is: 5′-TAGCCAAACGTCACCCAGAG-3′.

8. A combination of microbial markers for diagnosing or predicting healthy youth and healthy aging in humans, characterized in that: It is the intestinal bacteria Gordonibacter and / or C.scindens.

9. A system for predicting healthy youth and healthy aging of a human body, characterized in that: Includes the following modules: A data input module is used to input the abundance data of the microbial marker combination in the biological sample of the subject obtained, The microbial marker combination is intestinal bacteria Gordonibacter and / or C.scindens; The prediction module is connected to the data input module, sets the boundaries of healthy youth and healthy aging indicators of Gordonibacter and C.scindens, and predicts whether the subject is healthy young or healthy aging based on the abundance data of the microbial marker combination of the subject obtained from the data input module.

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