CN112852983A - Method for collecting intestinal flora and application thereof - Google Patents
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Abstract
The invention discloses a collection method of intestinal flora and application thereof, belonging to the field of medicine. The method comprises the following steps: (1) waiting for emptying the intestinal tract of the object collecting the intestinal tract flora to prepare the intestinal tract; (2) an endoscopist lavages colonic mucosa by using medical purified water through an endoscope image system and sucks the lavage liquid into a disposable sterile negative pressure drainage bag; (3) and centrifuging the lavage fluid in the negative pressure drainage bag, and collecting the precipitate, wherein the precipitate is the collected intestinal flora. The invention can be used for more accurately detecting and analyzing the abundance of the intestinal microorganisms, and the analysis result can better reflect the change condition of the intestinal flora in a disease state. Meanwhile, the method has the advantage of no wound.
Description
Technical Field
The invention relates to a collection method of intestinal flora and application thereof, belonging to the field of medicine.
Background
The intestinal flora is normal microorganisms in human intestinal tract, and mainly comprises bacteria, archaea, fungi, viruses and the like. The total number of intestinal bacteria, which is 100 trillion and 10 times the total number of human cells, affects body weight and digestive ability, protects against infection and risk of autoimmune disease, and controls the response of the human body to cancer therapeutic drugs.
The human body can be considered as "walking micro-organisms". Causal links between gut flora disturbances and abnormalities in human health are widely documented, and correlations between abundance of specific gut flora members and various diseases have also been discovered. The specific disordered bacteria can be the 'destroyer' of the intestinal function, and after the balance of the composition and diversity of the intestinal flora is broken, the intestinal micro-ecological disorder can be caused, so that the metabolic activity and the immune response of the intestinal flora are shifted, and the serious physiological or pathological consequences of the organism can be caused, therefore, the sampling detection and analysis of the change of the intestinal microorganisms have important significance on the health of the human body.
Stool is commonly used for non-invasive intestinal sampling to assess intestinal microbiota. However, stool samples are self-sampling, and the complexity of the participants, uncertainty in the sampling location or sampling time, all contribute to large errors. At present, the extraction of the intestinal mucosa microorganisms is completed by collecting the intestinal mucosa through biopsy forceps, and the sampling method has certain invasiveness and damages to the bodies of participants.
Colonoscopy is a commonly used fiberscope in clinic. The rectum, sigmoid colon, descending colon, transverse colon, ascending colon and cecum, as well as a small segment of the small intestine (ileocecal end) connected to the large intestine, can be examined by inserting the retrograde down through the anus. Intestinal lesions can be clearly found, and partial intestinal lesions can be treated, such as: directly remove benign lesions such as large intestine polyp under the microscope, stop bleeding of intestinal tract bleeding under the microscope, and remove foreign matters in the large intestine. During colonoscopy, physicians often wash the intestines with purified medical water to wash out substances that may interfere with the examination that remain in the intestines after they are prepared. Lavage fluids are typically disposed of as medical waste.
Disclosure of Invention
[ problem ] to
The existing method for sampling the intestinal microorganisms has certain defects, wherein the error of sampling the excrement is large, the analysis result is unstable and reliable, and the biopsy forceps have invasive sampling and harm to human bodies.
[ solution ]
The invention provides a non-invasive intestinal flora collection method, which comprises the following steps:
(1) waiting for emptying the intestinal tract of the object collecting the intestinal tract flora to prepare the intestinal tract;
(2) an endoscopist lavages colonic mucosa by using medical purified water through an endoscope image system and sucks the lavage liquid into a disposable sterile negative pressure drainage bag;
(3) and centrifuging the lavage fluid in the negative pressure drainage bag, and collecting the precipitate, wherein the precipitate is the collected intestinal flora.
Preferably, the intestinal tract preparation quality assessment is measured on the boston gut preparation scale (BBPS). The mucosa lavage fluid sample with the score of more than or equal to 6 on the Boston intestine preparation scale is a qualified and available sample.
Preferably, the lavage fluid in the negative pressure drainage bag is centrifuged at 9000rpm for 10min to collect the precipitate as an intestinal flora sample (mucosal lavage fluid sample).
The invention provides a non-invasive method for detecting intestinal flora, which comprises the following steps:
(1) centrifuging intestinal lavage fluid, and collecting precipitate, wherein the precipitate is intestinal flora sample;
(2) extracting genome of the intestinal flora sample, amplifying a V3-V4 segment of ribosome 16S rRNA, and performing high-throughput sequencing on the amplified segment to obtain the bacterial composition of the sample.
Preferably, the Boston bowel preparation scale score for intestinal lavage fluid is greater than or equal to 6.
The invention provides a non-invasive method for analyzing intestinal flora, which comprises the following steps:
(1) centrifuging intestinal lavage fluid, and collecting precipitate, wherein the precipitate is intestinal flora sample;
(2) extracting genome of the intestinal flora sample, amplifying a V3-V4 region segment of ribosome 16S rRNA, and performing high-throughput sequencing on the amplified segment;
(3) and (5) performing bioinformatics analysis on the sequencing result.
Preferably, the bioinformatic analysis is a microbial community diversity analysis of different samples, a principal component analysis of different samples, a compositional difference analysis of different samples at the phylum taxonomic level.
Preferably, the microbial community diversity analysis results are expressed in terms of species diversity index and species abundance.
[ advantageous effects ]
In the course of research, the inventors of the present invention have found that mucosal lavage fluid also contains certain intestinal microorganisms when performing a colorectal endoscopy. Therefore, when an endoscopist performs colonoscopy, the medical purified water is used for irrigating the colonic mucosa and collecting the irrigating solution, the irrigating solution can be used for more accurately detecting and analyzing the abundance of the intestinal microorganisms, and the analysis result can better reflect the change condition of the intestinal flora under the disease state.
The intestinal microorganism collection method provided by the invention also has the advantage of no wound.
Drawings
FIG. 1 shows the sampling process of the stool sample and the mucosal lavage fluid sample of example 1.
FIG. 2 is a graph showing comparison of electrophoresis detection of the V3-V4 sections of the mucosal lavage fluid and stool sample in example 1, wherein M in the leftmost column represents Marker, CK represents sterilized water, Y1 represents purified medical water, and 1-91 represent mucosal lavage fluid and stool sample.
FIG. 3 is a graph showing the comparison of the microbiological analysis of the mucosal lavage fluid and the fecal sample of example 1, (1) the fecal sample and mucosal lavage fluid sample of the healthy control group, (2) the fecal sample and mucosal lavage fluid sample of the polyp case, (3) the fecal sample and mucosal lavage fluid sample of the adenoma case, (4) the fecal sample of the healthy control group, the adenoma group, and (5) the mucosal lavage fluid sample of the healthy control group, the adenoma group.
FIG. 4 is a comparison of the microbiota classification levels of mucosal lavage fluid of example 1 with stool samples.
Detailed Description
Example 1
During the period from 12 months in 2018 to 6 months in 2019, a total of 62 standard-compliant volunteers, 22 healthy controls and 40 adenomas, were collected based on screening criteria and were enrolled in affiliated hospitals at southern Jiangnan university in Wuxi, a volunteer aged 18 years or older and not treated with antibiotics.
TABLE 1 volunteers' basic information and polyp characteristics
Colorectal polyps were pathologically confirmed by a colorectal scopy agent. The healthy control groups were: polyps were not found in the colorectal endoscopy, both in patients without a history of colorectal polyps and without other colorectal lesions. Polyp cases were defined as: there was at least one hyperplastic or inflammatory polyp, no other adenomatous polyp, or no histological classification. Cases of adenomas are defined as: there is at least one tubular adenoma, villous adenoma, tubular villous adenoma, or non-fixed serrated adenoma, and no other hyperplastic polyps or histological unclassification.
As shown in fig. 1, the stool sample and mucosal lavage sample collection procedure is illustrated. Wherein, the fecal sample is collected from the middle section of the fecal sample before intestinal tract preparation by volunteers, and the sample is frozen and stored at-80 ℃. After collecting the fecal samples, the volunteers took 3 boxes of laxatives (each box containing 59.0g of polyethylene glycol, 5.68g of sodium sulfate, 1.68g of sodium bicarbonate, 1.46g of sodium chloride, and 0.74g of potassium chloride) for bowel preparation. The following day, collection of mucosal lavage samples was performed: endoscopists performed colonoscopy on volunteers using a digital electronic endoscopic imaging system (Japanese Pantex EKP-150C), during which the endoscopist irrigated the colonic mucosa with 100mL of medical purified water and immediately aspirated the irrigation solution into a disposable sterile negative pressure drainage bag. After the colonoscopy is finished, collecting the effusion in the sterile negative-pressure drainage bag, centrifuging the effusion at a high speed (9000r/min), collecting the precipitate to obtain a mucosa lavage fluid sample, and freezing and storing the sample at the temperature of minus 80 ℃. Intestinal preparation quality evaluation is measured by a Boston intestinal preparation Scale (BBPS), and mucosal lavage fluid samples with the total BBPS score of more than or equal to 6 are selected for subsequent microbial flora analysis.
Extracting genome of the sample collected by the 2 modes, amplifying the ribosome 16S rRNA gene V3-V4 region fragment, performing agarose electrophoresis on the amplified fragment for gel cutting and purification, and sequencing by using an Illumina MiSeq sequencing platform in a PE300 mode. FIG. 2 shows the PCR amplification electrophoresis results of the 16S rRNA V3-V4 region fragment in the genome of the mucosal lavage fluid and the fecal sample, wherein the mucosal lavage fluid and the fecal sample both have a band around 250bp, which indicates that both the mucosal lavage fluid and the fecal sample contain bacteria.
And analyzing and comparing the sequencing results. Fecal samples and mucosal lavage samples from 62 volunteers were obtained, the results of the sequence determination of the microbial community diversity were obtained, and bioinformatics analysis was performed, and the results of the microbial community diversity analysis of each sample were expressed by the species diversity index (Shannon index) and the species abundance (i.e., the number of species in terms of the operational taxa, the number of OUT). The Shannon index curve of the sample is saturated, which shows that new species cannot be added along with the increase of the sequencing quantity, the diversity of the microorganisms cannot be changed, and the current sequencing quantity meets the experiment. In the experiment for researching the composition of the mucosa lavage fluid flora, 1265446 high-quality V4 sequences of 16SrRNA are generated in total through MiSeq high-throughput sequencing, 10606 sequences are averagely generated in each sample, the left sequence and the right sequence are clustered according to a reference sequence, OUT is divided according to 97% sequence similarity, and 123624 OUT is generated in total.
And calculating weighted UniFrace distances among different samples through bioinformatics analysis, performing principal component analysis and comparing. As shown in fig. 3, PCoA based on weighted UniFrac distance showed that the fecal sample and mucosal lavage sample data points were well separated on the first axis (PC 1). In cases of colorectal adenomas, the difference between mucosal lavage fluid samples was slightly higher than that of stool samples. Overall, two non-invasive sampling types from the same volunteer showed different microbiota, reflecting the more general advantage of mucosal lavage specimen type in the gut microbiota of disease.
The compositional differences of different samples at the phylum of bacteria classification level were compared by bioinformatics analysis and comparison. As shown in FIG. 4, the bacteria having a higher content in the intestinal flora were, in order, Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria in all volunteers. Fusobacteriales are enriched in the intestine of patients with colorectal adenomas and have been reported to be present in higher amounts in colorectal cancer tissues. Increased abundance of clostridia phyla in mucosal lavage fluid compared to fecal samples; for example, in the adenoma group, the abundance of the fusobacteria in the stool sample was only 2.2%, while the abundance of the fusobacteria in the mucosal lavage fluid sample was maintained at 11.7%, and the abundance of the fusobacteria in the adenoma group was higher than that in the healthy control group, the abundance of the fusobacteria in the mucosal lavage fluid of the healthy control group was 4.4%, and the abundance of the fusobacteria in the stool of the healthy control group was 0.4%.
By bioinformatic analysis and comparison, 30 abundantly different OTUs were identified in stool samples and mucosal lavage fluids, and 20 OTUs from haemophilus, clostridium, streptococcus, granulosum and other pathogens or opportunistic pathogens were increased in mucosal lavage fluids. And the difference between the mucosa lavage fluid sample and the feces sample is more obvious in the adenoma group compared with the healthy control group.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for non-invasive collection of gut flora comprising the steps of:
(1) waiting for emptying the intestinal tract of the object collecting the intestinal tract flora to prepare the intestinal tract;
(2) an endoscopist lavages colonic mucosa by using medical purified water through an endoscope image system and sucks the lavage liquid into a disposable sterile negative pressure drainage bag;
(3) and centrifuging the lavage fluid in the negative pressure drainage bag, and collecting the precipitate, wherein the precipitate is the collected intestinal flora.
2. The method of claim 1, wherein the evaluation of gut prep quality is measured on a boston gut prep scale.
3. The method of claim 1, wherein the lavage fluid in the negative pressure drainage bag is centrifuged at 9000rpm for 10min, and the precipitate is collected and precipitated as the intestinal flora sample.
4. A method for non-invasive detection of gut flora, comprising the steps of:
(1) centrifuging the intestinal lavage fluid to collect precipitate, wherein the precipitate is an intestinal flora sample;
(2) extracting genome of the intestinal flora sample, amplifying a V3-V4 segment of ribosome 16S rRNA, and performing high-throughput sequencing on the amplified segment to obtain the bacterial composition of the sample.
5. The method of claim 4, wherein the lavage fluid has a Boston bowel preparation scale score of 6 or more.
6. The method of claim 4, wherein the intestinal lavage fluid is centrifuged at 9000rpm for 10min to collect the pellet, which is then used as the intestinal flora sample.
7. A method for non-invasive analysis of gut flora, comprising the steps of:
(1) centrifuging the intestinal lavage fluid to collect precipitate, wherein the precipitate is an intestinal flora sample;
(2) extracting genome of the intestinal flora sample, amplifying a V3-V4 region segment of ribosome 16S rRNA, and performing high-throughput sequencing on the amplified segment;
(3) and (5) performing bioinformatics analysis on the sequencing result.
8. The method of claim 7, wherein the bioinformatic analysis is a microbiota diversity analysis of different samples, a principal component analysis of different samples, a compositional difference analysis of different samples at the phylum of bacteria classification level.
9. The method of claim 7, wherein the results of the microbiota diversity analysis are expressed in terms of species diversity index and species abundance.
10. The method of claim 7, wherein the intestinal lavage fluid is centrifuged at 9000rpm for 10min, and the pellet is collected and deposited as a sample of intestinal flora.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113913296A (en) * | 2021-10-11 | 2022-01-11 | 厦门承葛生物科技有限公司 | Centrifugal extraction method of intestinal flora |
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| CN113913296A (en) * | 2021-10-11 | 2022-01-11 | 厦门承葛生物科技有限公司 | Centrifugal extraction method of intestinal flora |
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