阅读说明：本技术 一种急性肺损伤肠道菌群多样性的分析方法 (Analysis method for diversity of intestinal flora in acute lung injury ) 是由 李志军 于 2020-12-14 设计创作，主要内容包括：本发明公开了一种急性肺损伤肠道菌群多样性的分析方法,涉及急性肺损伤肠道菌群多样性的分析技术领域,具体为一种急性肺损伤肠道菌群多样性的分析方法,包括以下步骤：S1、收集样本；S2、样本DNA抽提；S3、PCR扩增；S4、数据信息分析：S401、有效序列统计；S402、有效序列长度分布统计；S403、OUT聚类及物种信息标注；S404、多样性指数；S405、宏基因组测序分析。该急性肺损伤肠道菌群多样性的分析方法通过对急性肺损伤肠道菌群多样性能够有效的得到病患的详细具体数值,与标准数据值对比,能够有效的判断出病患肺部的详细情况,以及可开具详细表格备案,从而能够有效的根据病患肺部情况针对性治疗。(The invention discloses an analysis method of acute lung injury intestinal flora diversity, which relates to the technical field of analysis of acute lung injury intestinal flora diversity, in particular to an analysis method of acute lung injury intestinal flora diversity, and comprises the following steps: s1, collecting a sample; s2, extracting sample DNA; s3, PCR amplification; s4, analyzing data information: s401, counting effective sequences; s402, counting the length distribution of the effective sequences; s403, OUT clustering and species information labeling; s404, diversity index; s405, metagenome sequencing analysis. According to the analysis method for the diversity of the intestinal flora with acute lung injury, the detailed specific numerical values of the patient can be effectively obtained through the diversity of the intestinal flora with acute lung injury, the detailed conditions of the lung of the patient can be effectively judged by comparing the detailed numerical values with the standard data values, and a detailed table can be developed for record, so that the targeted treatment can be effectively carried out according to the lung conditions of the patient.)

1. An analysis method for the diversity of intestinal flora in acute lung injury comprises the following steps:

s1, collecting samples: excreting feces of a patient with acute lung injury into a clean container, sticking a small amount of feces with a cotton swab, placing the feces into a test tube containing sterile normal saline, covering the test tube with a cover for sealing, placing the test tube into a medical refrigerator, and refrigerating for 24 hours to stabilize flora without change;

s2, sample DNA extraction: conveying the test tube in the collected sample obtained in the step S1 to a sample extraction position, and extracting a DNA template;

s3, PCR amplification: extracting a bacterial genome DNA template in the step S2 of sample DNA extraction, wherein a PCR product comprises a region to be detected, a sequencing primer, a specific primer, a Barcode for distinguishing a sample and a sequencer, the specific primer is used for amplifying a V4 region of 16srDNA, agarose gel electrophoresis is adopted to detect the purity and specificity of the product after the sample is amplified, a clear and obvious main band is required to appear at the position of the corresponding fragment size, no obvious miscellaneous band is required at other positions, accurate quantification and purification recovery are carried out, sequencing of a sample is carried out according to the use instruction of the instrument, and data are generated.

S4, analyzing data information:

s401, counting effective sequences;

s402, counting the length distribution of the effective sequences;

s403, OUT clustering and species information labeling;

s404, diversity index;

s405, metagenome sequencing analysis.

2. The method for analyzing the intestinal flora diversity of acute lung injury according to claim 1, wherein: and S1, collecting the sample, wherein the sterile normal saline is 80-100 ml.

3. The method for analyzing the intestinal flora diversity of acute lung injury according to claim 1, wherein: s2, in the sample DNA extraction, selecting a single bacterium, dropping the single bacterium into a PA bottle containing 5-10mL of LB and MRS liquid culture medium, activating for 3-4h at 30 ℃, if the culture solution is slightly turbid, adding 1-2mL of the single bacterium into a sterilized centrifuge tube, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 1-2mLSTE to suspend the bacterium, repeating the steps, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 100 mu LTE to the collected bacterium, boiling for 5-15min in boiling water, immediately ice-cooling for 5-15min, centrifuging for 2-7min, taking the supernatant as a DNA template, placing the genome DNA at-20 ℃, and storing for later use.

4. The method for analyzing the bacterial population diversity of intestinal tract with acute lung injury according to claim 1, wherein in the step S3, PCR amplification, the amplicon sequence 16rDNA analysis process comprises a first step of sequencing data of the original amplicon; secondly, controlling and screening the data quality; thirdly, amplifying the sub-database for comparison; step four, OUT classification, species distribution and phylogenetic tree construction are respectively carried OUT; fifthly, carrying OUT biological species and abundance estimation on OUT classification and species distribution in the fourth step; sixthly, extracting multiple samples, and comparing community structures among the samples; seventhly, analyzing the microbial distribution and the environment.

5. The method for analyzing the intestinal flora diversity of acute lung injury according to claim 1, wherein: in the step S404, in the diversity index, the types, the quantities and the proportions of all bacteria are measured and used for determining the health grade condition of the human flora of the sample; and comparing the obtained data with a standard database, determining the similarity of the sample with healthy people and disease people, finally evaluating the health condition of the sample examined person, and performing post-processing estimation or hospitalizing treatment.

6. The method for analyzing the intestinal flora diversity of acute lung injury according to claim 1, wherein: in the step S405, in metagenomic sequencing analysis, correlation analysis is performed on the intestinal microorganisms and the diseases by a metagenomic sequencing method to reveal the microbial differences between the diseases and the healthy individuals; identifying specific microorganisms in specific environment to find tolerant strains and related genes; the classification of species, the study of metabolic pathways associated with a particular environment, and the study of relationships within microbial communities, between microorganisms and environments, and between microorganisms and hosts through comparison of different samples can be studied.

Technical Field

The invention relates to the technical field of analysis of diversity of intestinal flora caused by acute lung injury, in particular to an analysis method of diversity of intestinal flora caused by acute lung injury.

Background

Acute lung injury is the damage of alveolar epithelial cells and capillary endothelial cells caused by various direct and indirect injury factors, and causes diffuse pulmonary interstitial and alveolar edema, thereby causing acute hypoxic respiratory insufficiency. Pathophysiological features of reduced lung volume, reduced lung compliance, dysregulation of the ventilation/blood flow ratio, clinically manifested by progressive hypoxemia and respiratory distress, and pulmonary imaging manifested by non-uniform exudative lesions, whose progression to a severe stage (oxygenation index <200) is known as acute respiratory distress syndrome.

In the intestinal micro-ecological environment, 100 trillion microbes such as bacteria, fungi, viruses, protozoa and the like exist, wherein more than 100 kinds of microbes are bacteria and are collectively called intestinal flora. They have the functions of synthesizing vitamins and proteins, activating immunity, resisting tumor, detoxifying and cleaning the internal environment of the intestinal tract. Generally, the intestinal flora is in a state of equilibrium with the human body and the external environment, which plays an important role in the health of the human body, but the equilibrium can be broken under certain conditions to form intestinal flora imbalance, which is represented by changes in the species, quantity, proportion, location and biological characteristics of the intestinal flora.

The existing analysis of the diversity of the intestinal flora is not targeted, and data errors are easy to generate, so that the defect that the judgment precision of acute lung injury of a patient is reduced is caused.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an analysis method for the diversity of intestinal flora caused by acute lung injury, which solves the problem that the existing analysis for the diversity of the intestinal flora in the background art is not targeted and is easy to generate data errors, so that the judgment precision of acute lung injury of a patient is reduced.

In order to achieve the purpose, the invention is realized by the following technical scheme: an analysis method for the diversity of intestinal flora in acute lung injury comprises the following steps:

s1, collecting samples: excreting feces of a patient with acute lung injury into a clean container, sticking a small amount of feces with a cotton swab, placing the feces into a test tube containing sterile normal saline, covering the test tube with a cover for sealing, placing the test tube into a medical refrigerator, and refrigerating for 24 hours to stabilize flora without change;

s2, sample DNA extraction: conveying the test tube in the collected sample obtained in the step S1 to a sample extraction position, and extracting a DNA template;

s3, PCR amplification: extracting a bacterial genome DNA template in the step S2 of sample DNA extraction, wherein a PCR product comprises a region to be detected, a sequencing primer, a specific primer, a Barcode for distinguishing a sample and a sequencer, the specific primer is used for amplifying a V4 region of 16srDNA, agarose gel electrophoresis is adopted to detect the purity and specificity of the product after the sample is amplified, a clear and obvious main band is required to appear at the position of the corresponding fragment size, no obvious miscellaneous band is required at other positions, accurate quantification and purification recovery are carried out, sequencing of a sample is carried out according to the use instruction of the instrument, and data are generated.

S4, analyzing data information:

s401, counting effective sequences;

s402, counting the length distribution of the effective sequences;

s403, OUT clustering and species information labeling;

s404, diversity index;

s405, metagenome sequencing analysis.

Optionally, in step S1, in the collected sample, the sterile physiological saline is 80-100 ml.

Optionally, in the step S2, in the sample DNA extraction, selecting a single bacterium, dropping the single bacterium into 5-10mL of PA bottle containing LB and MRS liquid culture medium, activating for 3-4 hours at 30 ℃, if the culture solution is slightly turbid, adding 1-2mL of the single bacterium into a sterilized centrifuge tube, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 1-2mL of tste to suspend the bacterium, repeating the above steps, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 100 mu LTE to the collected bacterium, boiling for 5-15min in boiling water, immediately ice-cooling for 5-15min, centrifuging for 2-7min, taking the supernatant as a DNA template, placing the genomic DNA at-20 ℃, and storing for later use.

Optionally, in the step S3, in PCR amplification, the amplicon sequence 16rDNA analysis process, the first step, the original amplicon sequencing data; secondly, controlling and screening the data quality; thirdly, amplifying the sub-database for comparison; step four, OUT classification, species distribution and phylogenetic tree construction are respectively carried OUT; fifthly, carrying OUT biological species and abundance estimation on OUT classification and species distribution in the fourth step; sixthly, extracting multiple samples, and comparing community structures among the samples; seventhly, analyzing the microbial distribution and the environment.

Optionally, in the step S404, in the diversity index, the types, the numbers and the proportions of all bacteria are measured, and are used for determining the health level condition of the sample human flora; and comparing the obtained data with a standard database, determining the similarity of the sample with healthy people and disease people, finally evaluating the health condition of the sample examined person, and performing post-processing estimation or hospitalizing treatment.

Optionally, in the step S405, in the metagenome sequencing analysis, the correlation analysis is performed on the intestinal microorganisms and the disease by using a metagenome sequencing method to reveal the microbial difference between the disease and the healthy individual; identifying specific microorganisms in specific environment to find tolerant strains and related genes; the classification of species, the study of metabolic pathways associated with a particular environment, and the study of relationships within microbial communities, between microorganisms and environments, and between microorganisms and hosts through comparison of different samples can be studied.

The invention provides an analysis method for the diversity of intestinal flora in acute lung injury, which has the following beneficial effects:

the analysis method for the diversity of the acute lung injury intestinal flora comprises the steps of obtaining detailed specific numerical values of patients by effectively obtaining the diversity of the acute lung injury intestinal flora, comparing the detailed numerical values with standard data values, effectively judging the detailed conditions of the lungs of the patients, and developing detailed form records, thereby being capable of effectively treating the lungs of the patients according to the pertinence of the lung conditions of the patients, judging the influence of treatment on the lungs of the patients, comparing the lung data records of the patients with the lung data forms issued at every time, effectively judging the recovery conditions of the lungs of the patients, effectively improving the treatment effect of the lungs of the patients, developing consultation and effectively improving the treatment success rate.

Detailed Description

In the following, technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.

The invention provides a technical scheme that: an analysis method for the diversity of intestinal flora in acute lung injury comprises the following steps:

s1, collecting samples: excreting feces of a patient with acute lung injury into a clean container, sticking a small amount of feces with a cotton swab, placing the feces into a test tube containing sterile normal saline, covering the test tube with a cover for sealing, placing the test tube into a medical refrigerator, and refrigerating for 24 hours to stabilize flora without change;

s2, sample DNA extraction: conveying the test tube in the collected sample obtained in the step S1 to a sample extraction position, and extracting a DNA template;

s3, PCR amplification: extracting a bacterial genome DNA template in the step S2 of sample DNA extraction, wherein a PCR product comprises a region to be detected, a sequencing primer, a specific primer, a Barcode for distinguishing a sample and a sequencer, the specific primer is used for amplifying a V4 region of 16srDNA, agarose gel electrophoresis is adopted to detect the purity and specificity of the product after the sample is amplified, a clear and obvious main band is required to appear at the position of the corresponding fragment size, no obvious miscellaneous band is required at other positions, accurate quantification and purification recovery are carried out, sequencing of a sample is carried out according to the use instruction of the instrument, and data are generated.

S4, analyzing data information:

s401, counting effective sequences;

s402, counting the length distribution of the effective sequences;

s403, OUT clustering and species information labeling;

s404, diversity index;

s405, metagenome sequencing analysis.

In the invention: and step S1, collecting the sample, wherein the sterile normal saline is 80-100 ml.

In the invention: s2, in sample DNA extraction, selecting a single bacterium, dropping the single bacterium into a PA bottle containing 5-10mL of LB and MRS liquid culture medium, activating for 3-4h at 30 ℃, if the culture solution is slightly turbid, adding 1-2mL of the single bacterium into a sterilized centrifuge tube, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 1-2mLSTE to suspend the bacterium, repeating the steps, centrifuging for 1-3min at 12000r/min, removing the supernatant, adding 100 mu LTE to the collected bacterium, boiling in boiling water for 5-15min, immediately ice-bathing for 5-15min, centrifuging for 2-7min, taking the supernatant as a DNA template, placing the genome DNA at-20 ℃ and storing for later use.

In the invention: s3, in PCR amplification, the amplicon sequence 16rDNA analysis process, the first step, the sequencing data of the original amplicon; secondly, controlling and screening the data quality; thirdly, amplifying the sub-database for comparison; step four, OUT classification, species distribution and phylogenetic tree construction are respectively carried OUT; fifthly, carrying OUT biological species and abundance estimation on OUT classification and species distribution in the fourth step; sixthly, extracting multiple samples, and comparing community structures among the samples; seventhly, analyzing the microbial distribution and the environment.

In the invention: step S404, in the diversity index, measuring the types, the quantities and the proportions of all bacteria, and determining the health grade condition of the human flora of the sample; and comparing the obtained data with a standard database, determining the similarity of the sample with healthy people and disease people, finally evaluating the health condition of the sample examined person, and performing post-processing estimation or hospitalizing treatment.

In the invention: step S405, in the metagenome sequencing analysis, performing correlation analysis on the intestinal microorganisms and the diseases by using a metagenome sequencing method to reveal the microbial difference between the diseases and the healthy individuals; identifying specific microorganisms in specific environment to find tolerant strains and related genes; the classification of species, the study of metabolic pathways associated with a particular environment, and the study of relationships within microbial communities, between microorganisms and environments, and between microorganisms and hosts through comparison of different samples can be studied.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.