CN113549671B - Intestinal flora for diagnosing sarcopenia of maintenance hemodialysis patient - Google Patents

Abstract

The application discloses an intestinal flora for diagnosing sarcopenia of a patient with maintenance hemodialysis, wherein the intestinal flora at least comprises Megamonas; the relative abundance of Megamonas is significantly reduced (P < 0.001) in patients with MHD sarcopenia. The diagnostic value of Megamonas on MHD sarcopenia is expressed by ROC and AUC, the AUC is 0.787, the sensitivity is 0.867, the specificity is 0.800, and the diagnostic effect of Megamonas on MHD sarcopenia is higher.

Description

A gut microbiota for diagnosing sarcopenia in maintenance hemodialysis patients

Technical field

The invention belongs to the field of biotechnology, and specifically relates to an intestinal flora for diagnosing sarcopenia in maintenance hemodialysis patients.

Background technique

Sarcopenia is a group of degenerative syndromes characterized by reduced skeletal muscle mass, weakened muscle strength, and/or reduced physical function. Maintenance hemodialysis (MHD) patients are at high risk of developing sarcopenia due to negative protein balance caused by kidney disease, dialysis treatment, and chronic inflammation, as well as dietary restrictions and reduced physical activity. Sarcopenia not only reduces the physical function of MHD patients, impairs daily living activities, and leads to a decline in patients' quality of life, but also increases the risk of hospitalization and hospitalization costs, and is even an independent predictor of mortality in MHD patients, bringing greater harm to families and society. burden.

Intestinal flora can produce a variety of metabolites that affect human health, thereby affecting the health status of the host. Previous studies have believed that the intestinal flora affects the host's muscle mass and function by directly affecting the host's inflammatory environment, nutrient bioavailability, lipid metabolism and energy supply. However, it is mainly based on animal models, and human studies are limited and have no The study directly explores the characteristics of the gut microbiota in people with MHD sarcopenia. Studying the characteristics of the intestinal flora in patients with MHD sarcopenia and identifying the marker bacterial genera related to sarcopenia is of great significance for revealing the pathogenesis of MHD sarcopenia and realizing the diagnosis and treatment of sarcopenia.

Contents of the invention

The purpose of the present invention is to provide intestinal flora related to the occurrence and development of sarcopenia in hemodialysis patients and to use it for diagnosing and treating sarcopenia in hemodialysis patients.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

An intestinal flora for diagnosing sarcopenia in maintenance hemodialysis patients, wherein the intestinal flora at least includes Megamonas.

A method for screening the intestinal flora of sarcopenia in maintenance hemodialysis patients, the method is as follows:

Step 1: Collect fresh feces from hemodialysis patients with sarcopenia and hemodialysis patients without sarcopenia, and immediately store them at -80°C for examination after sampling;

Step 2: 16S rRNA sequencing, extract genomic DNA from the sample, and use agarose gel electrophoresis to detect the quality of genomic DNA;

Step 3: Use primers 338F and 806R to PCR amplify the V3-V4 variable region of bacterial 16S rRNA; the PCR products are mixed in equal proportions and then purified using a DNA purification column;

Step 4: Construct a library from the purified amplified fragments according to the standard operating procedures of the IlluminaMiSeq platform; use the Hiseq2500 platform for sequencing;

Step 5: Use FLASH 1.2.11 software to splice the original sequencing sequences, use Trimmomatic 0.33 software for quality filtering, and use UCHIME 8.1 software to remove chimeras to obtain high-quality Tags sequences;

Step 6: Use USEARCH 10.0 software to perform OTU clustering on the sequences at the 97% level of similarity, and remove single sequences and chimeras during the clustering process; use the RDP classifier to perform OTU clustering at the 97% level for biological information. Statistical analysis, using Silva database (SSU123) for comparison;

Step 7: Use the Kruskal-Wallis rank sum test of SPSS 26.0 software to analyze the differential bacterial flora between the groups. The differential bacterial flora between the groups is Megamonas, and use the ROC curve and the area under the curve to evaluate the effect of the differential bacterial species on MHD muscle. Diagnostic value of oligophobia.

Preferably, 1.8% agarose gel electrophoresis is used to detect the quality of genomic DNA, and the quality of DNA includes the concentration and purity of DNA.

Preferably, the target nucleotide sequence of Megamonas is a fragment of the 16S rRNA gene.

A kit for predicting maintenance hemodialysis sarcopenia, the kit includes a reagent for detecting the abundance of Megamonas.

A composition and its use in preparing a medicament for treating maintenance hemodialysis sarcopenia, the composition includes an agent that increases the abundance of Megamonas.

The above technical solution can achieve the following beneficial effects:

The relative abundance of Megamonas in patients with maintenance hemodialysis sarcopenia of the present invention is significantly reduced (P<0.001). The diagnostic value of Megamonas bacteria for MHD sarcopenia was expressed by ROC and AUC. The AUC was 0.787, the sensitivity was 0.867, and the specificity was 0.800, indicating that Megamonas has a high diagnostic efficiency for MHD sarcopenia.

Description of the drawings

Figure 1 shows the species diversity analysis.

Figure 2 is a difference diagram of Megamonas.

Figure 3 is the ROC curve of Megamonas in diagnosing MHD sarcopenia.

Figure 4 is the ROC curve of Eisenbergiella in diagnosing MHD sarcopenia.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings:

Example 1: Screening intestinal flora related to sarcopenia:

1. Research subjects and sample collection: Collect fresh feces from the center of the feces of 30 MHD sarcopenia patients and 30 MHD non-sarcopenia patients, and immediately store them at -80°C for examination after sampling. The general information of the patients is shown in Table 1.

Table 1 General information of patients

feature Sarcopenia (n=30) Non-sarcopenia (n=30) age 49.9±12.6 45.87±12.3 Sex: Male) 17(56.7%) 17(56.7%) Dialysis age 36(24.75,76.25) 35.5(16.5,65.25) KT/V 1.53±0.31 1.43±0.29 Height(cm) 162.13±8.28 167.68±8.43 Weight(kg) 52.35±7.74 68.30±14.97 BMI(kg/m2) 19.93±2.70 24.12±3.94 SMI(kg/m2) 6.07±0.86 8.27±1.60 Grip strength(kg) 27.15(21.25,34.20) 32.25(27.05,44.60) Pace(m/s) 0.76±0.24 0.91±0.12

2.16S rRNA sequencing:

2.1 Extract DNA:

Genomic DNA was extracted from the sample according to the kit instructions.

2.2 Determine the concentration and purity of NDA:

Use 1.8% agarose gel electrophoresis to detect the quality of genomic DNA, including DNA concentration and purity

2.3PCR amplification and product purification:

The V3-V4 variable region of bacterial 16S rRNA was PCR amplified using primers 338F and 806R. The PCR products were mixed in equal proportions and then purified using a DNA purification column.

2.4Miseq library construction and sequencing:

A library was constructed from the purified amplified fragments according to the standard operating procedures of the Illumina MiSeq platform. Sequencing was performed using Illumina's Hiseq 2500 platform.

3. Data analysis:

3.1 Sequence splicing and filtering

Use FLASH 1.2.11 software to splice the original sequencing sequences, use Trimmomatic 0.33 software for quality filtering, and use UCHIME 8.1 software to remove chimeras to obtain high-quality Tags sequences.

3.2OTU clustering

Use USEARCH 10.0 software to perform OTU clustering on the sequences at a similarity level of 97%, and remove single sequences and chimeras during the clustering process. The OTUs of the RDP classifier at the 97% level were used for statistical analysis of bioinformatics, and the Silva database (SSU123) was used for comparison, where OTU represents operational taxonomic units.

3.3 Analysis of differences in intestinal flora species:

The Kruskal-Wallis rank sum test of SPSS 26.0 software was used to analyze the differential bacterial flora between groups, and the ROC curve and the area under the curve were used to evaluate the diagnostic value of differential bacterial genera for MHD sarcopenia.

4. Result:

The sequencing data and OTU information are shown in Table 2. A represents the MHD sarcopenia group, and B represents the MHD non-sarcopenia group. Species diversity analysis is shown in Figure 1. The species richness is reflected by the length of the curve on the horizontal axis. The wider the curve, the richer the species composition. The evenness of species composition is reflected by the shape of the curve. The longer the curve, the richer the species composition. Flat means the species composition is more uniform.

Table 2 Sequencing data and OTU number statistics

The results of species difference analysis showed that compared with MHD non-sarcopenia patients, the relative abundance of Megamonas in patients with MHD sarcopenia was significantly reduced (P<0.001). The difference is shown in Figure 2.

Example 2: Verification of the diagnostic value of Megamonas for MHD sarcopenia:

A: Research subjects and sample collection: Collect fresh feces from the center of the feces of 30 MHD sarcopenia patients and 30 MHD non-sarcopenia patients according to the method in Example 1. Immediately after sampling, store at -80°C for examination. The general information of the patients is shown in Table 3.

Table 3 General information of patients

feature Sarcopenia (n=30) Non-sarcopenia (n=30) age 50.1±10.6 49.3±9.6 Sex: Male) 17(56.7%) 17(56.7%) Dialysis age 39(27.25,79.75) 36.5(17.5,66.5) KT/V 1.74±0.35 1.65±0.33 Height(cm) 160.24±7.36 165.67±6.52 Weight(kg) 51.34±5.37 65.39±18.85 BMI(kg/m2) 19.92±2.45 23.87±3.56 SMI(kg/m2) 5.65±0.78 7.89±1.27 Grip strength (kg) 26.54(20.45,33.20) 31.38(26.93,42.63) Pace(m/s) 0.75±0.47 0.87±0.46

B.16S rRNA sequencing and data analysis are the same as in Example 1.

C. Result:

The test results showed that the relative abundance of Megamonas in maintenance hemodialysis patients was significantly reduced. The diagnostic value of Megamonas for MHD sarcopenia is represented by ROC and AUC, as shown in Figure 3. The AUC is 0.787, the sensitivity is 0.867, and the specificity is 0.800, indicating that Megamonas has a high diagnostic efficiency for MHD sarcopenia.

Among them: ROC represents the receiver operating curve; AUC represents the area under the curve.

Example 3:

Currently, Anaerofilum and Eisenbergiella bacteria are also used to diagnose or detect sarcopenia. When applying Anaerofilum and Eisenbergiella bacteria to the diagnosis of MHD sarcopenia, the inventors can conclude through experimental analysis that Anaerofilum bacteria are not present in the feces of MHD patients. The possible reason for the detection is that the relative abundance of Anaerofilum bacteria is too low and is not the dominant flora in the feces of MHD patients. Therefore, Anaerofilum bacteria cannot be used in the diagnosis of MHD sarcopenia.

The results of the detection of MHD sarcopenia by Eisenbergiella showed that the AUC in the ROC result was only 0.626 (see Figure 4 for details), the specificity was 0.567, and the sensitivity was 0.667. The diagnostic value is low and it cannot diagnose maintenance hemodialysis well. Sarcopenia.

The above are all preferred embodiments of the present invention. For those of ordinary skill in the art, without departing from the principles of the present invention, modifications to various equivalent forms of the present invention all fall within the scope of the appended claims of this application. protected range.

Claims (1)

1. Use of an agent for detecting the abundance of Megamonas in the intestinal flora for the preparation of a product for diagnosing sarcopenia in a patient suffering from maintenance hemodialysis, characterized in that: the relative abundance of Megamonas in patients with persistent hemodialysis sarcopenia is significantly reduced compared to patients with persistent hemodialysis non-sarcopenia.