Sample treatment method for intestinal flora detection

Abstract

The invention belongs to the technical field of microbial separation, and discloses a sample treatment method for intestinal flora detection, which comprises the following steps: collecting an intestinal flora sample by using a disposable sterile collection tool, transferring the sample into a sterile container, transferring the collected sample under a low-temperature condition, or storing the collected sample at normal temperature by using a stabilizer, and collecting the intestinal flora sample. And carrying out homogenization treatment on the sample, removing impurities, and subpackaging. According to the sample processing method for intestinal flora detection, high efficiency and traceability of sample collection and storage are achieved, a special sterile collection tool and a waterproof label are adopted, it is guaranteed that samples are free of exogenous pollution, information can be traced in the whole process, and the problems of sample confusion and information loss in a traditional method are solved; the preservation system is flexible and adaptive: through the accurate proportion of the preservation liquid and the temperature strategy, nuclease and flora metabolism is inhibited, limitation of professional cold chain equipment is avoided, and the method is suitable for clinical detection and large-scale epidemiological investigation.

Description

Technical Field

[0001] This invention relates to the field of microbial isolation technology, specifically to a sample processing method for detecting intestinal flora. Background Technology

[0002] In the fields of life sciences and medical research, the gut microbiota, as an important component of the human micro-ecosystem, is closely related to host processes such as nutrient metabolism, immune regulation, and disease development. Analysis of the nucleic acids (DNA / RNA) of the gut microbiota, such as 16S rRNA sequencing and metagenomics studies, can provide in-depth insights into the structure and function of the gut microbiota, offering crucial evidence for disease diagnosis, personalized medicine, and drug development. However, current techniques for gut microbiota sample processing and nucleic acid extraction still face many unresolved challenges. In the sample collection and preservation process, traditional methods struggle to balance microbial stability with ease of operation. On the one hand, intestinal samples such as feces have high microbial activity, and their microbial community structure is easily altered and nucleic acid degraded at room temperature, leading to sample distortion. Therefore, it is necessary to develop a standardized, efficient, and highly operable technical solution for gut microbiota sample processing and nucleic acid extraction to solve problems such as poor sample preservation stability, low nucleic acid extraction efficiency, and difficulty in ensuring quality, so as to provide more reliable technical support for gut microbiota research. Summary of the Invention

[0003] The purpose of this invention is to provide a sample processing method for intestinal flora detection, in order to solve the problem mentioned in the background art that the existing intestinal flora detection sample processing methods are difficult to balance microbial stability and operational convenience, and are prone to sample distortion.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a sample processing method for detecting intestinal flora, comprising: Intestinal flora samples were collected using disposable sterile collection tools and transferred to sterile containers. The collected samples were transported under low temperature conditions or stored at room temperature using stabilizers. The samples were homogenized, impurities were removed, and then aliquoted. Nucleic acid was extracted from gut microbiota samples to obtain complete microbial genomic DNA suitable for high-throughput sequencing.

[0005] Preferably, the sterile container is a 5mL screw-cap cryovial or an OMNIgene•GUT collection kit, with a waterproof label attached to the outside of the cryovial, indicating the sample ID, collection time, and clinical information.

[0006] By adopting the above technical solution, key information is marked with waterproof labels to link samples with patients, avoid confusion, and achieve traceable management of samples.

[0007] Preferably, the collection of the gut microbiota sample specifically includes: For hard stools, use a sterile spoon, wooden scraper or disposable stool collector to collect 2-3g samples from different areas inside the stool; For loose stools, use a disposable sterile spoon or sterile pipette to collect the stool.

[0008] By adopting the above technical solution, sterile disposable sample collectors are used to collect samples aseptically, thus avoiding sample contamination.

[0009] Preferably, when using the OMNIgene•GUT collection kit, the preservation solution is mixed with feces at a 1:1 volume ratio, vortexed for 10 seconds, and the mixed sample can be stably preserved for 14-30 days at room temperature of 18-25℃; the preservation solution is selected from any of the following: RNAlater is used to inhibit nuclease activity; 70%-95% ethanol is used to fix the bacterial community structure; PBS buffer containing antibiotics is used to inhibit bacterial growth; When RNAlater is used as the preservation solution, the final concentration should be ≥0.5% (v / v) to effectively inhibit nuclease activity. For short-term preservation (≤1 week), it can be carried out at room temperature. For long-term preservation, it needs to be transferred to 4℃ or -20℃. Processing within 48 hours does not require liquid nitrogen freezing.

[0010] By employing the above technical solution, through precise preservation solution ratios, standardized operations, and temperature strategies, stable preservation of intestinal microbial samples can be achieved without the need for complex equipment, providing a convenient and efficient sample processing solution for clinical microbiota detection, epidemiological surveys, and intestinal microecology research.

[0011] Preferably, if samples need to be frozen, they should be immediately placed in an environment of -80°C after collection, or briefly stored in an environment of -20°C and then transferred to -80°C, or stored in dry ice for a short period of time.

[0012] By employing the above technical solutions, through temperature gradient control, step-by-step operation, and material optimization, long-term stable maintenance of sample activity has been achieved in biobanks, clinical testing, and microbial research, providing a reliable sample basis for downstream experiments in molecular biology, microbiome, and other fields.

[0013] Preferably, the sample homogenization process adopts any of the following methods: Use a bead mill with parameters set to 6.0 m / s for 40-50 seconds. Manual vortex oscillation, time ≥ 2 minutes; Ultrasonic breaker, power 300W, working time 10-15 seconds / cycle, 30-second interval, 3-5 cycles; After homogenization, centrifuge at 3000-5000 rpm for 5 minutes, discard the supernatant, and the precipitate is the bacterial community enrichment.

[0014] By adopting the above technical solution, the release rate of microorganisms in intestinal samples is greatly improved through standardized homogenization and centrifugation enrichment.

[0015] Preferably, the nucleic acid extraction includes the following steps: 1) Take 200 μL of homogenized fecal suspension and add it to the lysis tube; 2) Add 800-1000 μL of PowerBead Solution buffer; 3) Add 80-100 μL of proteinase K, mix thoroughly, and then lyse to release DNA; Nucleic acid extraction from gut microbiota samples should be performed in a biosafety cabinet.

[0016] Using the above technical solution, nucleic acid extraction from intestinal flora samples is performed in a biosafety cabinet, achieving the dual requirements of biosafety protection and experimental quality control. Preferably, if the Firmicutes content in the sample is high, an additional 20-30 μL of lysozyme (10 mg / mL) should be added, and the solution should be incubated at 36-39°C for 30 minutes, followed by vortexing for 10-15 minutes until the solution is homogeneous, milky white, and free of visible particles.

[0017] Using the above technical solution, for samples with high Firmicutes content, lysozyme pretreatment combined with temperature-controlled incubation and vortex oscillation is a targeted enhanced lysis strategy. Through the dual action of "enzymatic destruction of the cell wall skeleton + mechanical disruption to release nucleic acids", the problem of Firmicutes being difficult to lyse is solved.

[0018] Preferably, the nucleic acid extraction further includes: 1) After lysis, centrifuge at below 4°C for 1-2 minutes and collect the supernatant; 2) Add 1-2 mL of Buffer C1 and vortex for 5-10 seconds; 3) Add the mixture to the silica gel column in portions, centrifuge for 1-2 minutes, and discard the waste liquid; 4) Add 500 μL of Buffer C2, centrifuge for 1 minute, and discard the waste liquid; 5) After washing, open the lid and let it sit for 2-3 minutes; 6) Elute with 60-80 μL of TE Buffer preheated to 60°C, let stand for 5-8 minutes, then centrifuge for 1-2 minutes to collect the DNA.

[0019] Using the above technical solution, the efficient extraction of intestinal flora DNA is achieved through a logical chain of "solid-liquid separation - impurity removal - nucleic acid adsorption - precise elution".

[0020] Preferably, the eluted DNA needs to undergo quality testing, including concentration testing and integrity testing.

[0021] Using the above technical solution, DNA quality detection is a key quality control step in the molecular analysis of gut microbiota.

[0022] Compared with the prior art, the beneficial effects of the present invention are: the sample processing method for detecting intestinal flora; 1. High efficiency and traceability of sample collection and preservation: Specialized sterile collection tools and waterproof labels are used to ensure that the samples are free from external contamination and that the information can be traced throughout the process, solving the problems of sample confusion and information loss in traditional methods. The preservation system is flexible and adaptable: Through precise ratio of preservation solution and temperature strategy, it inhibits nuclease and microbial metabolism, and is not limited by professional cold chain equipment, making it suitable for clinical testing and large-scale epidemiological surveys. 2. Precision and reliability of nucleic acid extraction quality control, targeted lysis and efficient enrichment: For difficult-to-lyse bacterial groups such as Firmicutes, lysozyme pretreatment combined with mechanical disruption is used to increase the microbial release rate to over 80%, solving the problem of bacterial abundance detection deviation. Purification system and quality control: Through silica column adsorption-washing-elution process and triple quality control, high-purity and complete DNA is obtained, ensuring the accuracy of high-throughput sequencing data. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the processing of intestinal flora detection samples according to the present invention. Figure 2 This is a comparison diagram of different sample preservation methods of the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see Figure 1-2 This invention provides a technical solution: a sample processing method for detecting intestinal flora, comprising: Intestinal flora samples were collected using disposable sterile collection tools and transferred to sterile containers. The sterile containers were 5mL screw-cap cryovials or OMNIgene•GUT collection kits. Waterproof labels were attached to the outside of the cryovials, marking the sample ID, collection time, and clinical information (subject's age, gender, disease, etc.). Background information such as dietary history, medication history, and sampling environment temperature were recorded simultaneously. The specific steps for collecting gut microbiota samples are as follows: During sampling, avoid direct hand contact with the sample. Wear sterile gloves and a mask. For hard stools, use a sterile spoon, wooden scraper, or disposable stool collector to collect 2-3g of sample from different areas inside the stool. Avoid urine during collection and avoid contact with the toilet bowl or toilet paper (if collecting intestinal fluid or intestinal biopsy samples, use a sterile syringe or tweezers to collect and quickly transfer to the preservation solution). The collection time should be within 15 minutes of stool expulsion (collect during natural defecation and avoid using enemas or other medications that may irritate the intestines). For loose stools, use a disposable sterile spoon or sterile pipette to collect the sample. The collected samples were transported under low temperature conditions or stored at room temperature using stabilizers. When using the OMNIgene•GUT collection kit, mix the preservation solution with the feces at a 1:1 volume ratio, vortex for 10 seconds, and the mixed sample can be stably stored for 14-30 days at room temperature (18-25℃). Choose any of the following preservation solutions: RNAlater is used to inhibit nuclease activity; 70%-95% ethanol is used to fix the bacterial community structure; PBS buffer containing antibiotics is used to inhibit bacterial growth; When RNAlater is used as the preservation solution, the final concentration should be ≥0.5% (v / v) to effectively inhibit nuclease activity. For short preservation periods (≤1 week), it can be carried out at room temperature. For long-term preservation, it needs to be transferred to 4℃ or -20℃. Processing within 48 hours does not require liquid nitrogen freezing. If samples need to be cryopreserved, they should be immediately placed in an environment of -80℃ after collection (they must be aliquoted and cryopreserved within 2 hours), or they can be temporarily stored in an environment of -20℃ and then transferred to -80℃. If long-term cryopreservation is required, samples should be stored in cryovials made of low-temperature resistant materials (preferably polyethylene) and glass containers should be avoided to prevent them from cracking.

[0026] Dry ice transport: suitable for cross-regional sample transfer, ensuring that the sample core temperature is ≤-60℃ during transport, using a temperature recorder to monitor temperature fluctuations during transport, and avoiding exceeding -20℃.

[0027] Sample transport packaging requirements: three-layer packaging, inner layer of sterile cryopreservation tube, middle layer of sealing leak-proof tape, and outer layer of insulated box or insulated dry ice bag.

[0028] The samples were homogenized, impurities were removed, and then aliquoted. Sample homogenization can be performed using any of the following methods: FastPrep-24 bead mill is preferred. Use a FastPrep-24 bead mill with parameters set to 6.0 m / s for 40-50 seconds (add 0.5g of zirconia beads (0.1mm + 0.5mm mixed) when using the bead mill), and cool in an ice bath for 5 minutes; Manual vortex oscillation, time ≥2 minutes, intermittent ice bath (avoid overheating); Ultrasonic breaker, power 300W, working time 10-15 seconds / cycle, 30-second interval, 3-5 cycles; After homogenization, centrifuge at 3000-5000 rpm for 5 minutes, discard the supernatant, and the precipitate is the microbial community enrichment. If the sample contains a lot of food residue, it needs to be filtered with sterile gauze or sterile filter. Before homogenization, samples need to be thawed: -80℃ samples are placed on ice and thawed slowly to avoid repeated freeze-thaw cycles, prevent damage to cell walls, and ensure nucleic acid yield. If solid fecal samples clump together, they need to be gently mixed using a sterile pipette or grinding rod, or vortexed with buffer solution (for preservation samples).

[0029] Nucleic acid is extracted from gut microbiota samples to obtain complete microbial genomic DNA suitable for high-throughput sequencing. Nucleic acid extraction includes the following steps: 1) Take 200 μL of homogenized fecal suspension and add it to a lysis tube. If the sample is dry, resuspend it in 100 μL of PBS first. 2) Add 800-1000 μL of PowerBead Solution buffer; 3) Add 80-100 μL of proteinase K, mix thoroughly, and then lyse to release DNA; Nucleic acid extraction from gut microbiota samples should be performed in a biosafety cabinet.

[0030] If the Firmicutes content in the sample is high, an additional 20-30 μL of lysozyme (10 mg / mL) should be added, and the solution should be incubated at 36-39°C for 30 minutes, followed by vortexing for 10-15 minutes until the solution is homogeneous, milky white, and free of visible particles.

[0031] Impurity precipitation and removal: 1) Centrifuge for 1 minute, 13000×g, temperature controlled at 4℃ (precipitate residue); 2) Transfer 700 μL to a new tube, avoiding the intake of precipitated impurities; 3) If the sample contains humic acid (A260 / A230<1.5), add 50μL of 25% PVP-40, incubate on ice for 10 minutes, and then centrifuge.

[0032] Nucleic acid extraction further includes: 1) After lysis, centrifuge at below 4°C for 1-2 minutes and collect the supernatant; 2) Add 1-2 mL of Buffer C1, vortex for 5-10 seconds to break hydrogen bonds and allow DNA to bind to the silica membrane; 3) Add the mixture to the silica gel column in portions, centrifuge for 1-2 minutes and discard the waste liquid to avoid excessive centrifugal force that could cause the membrane to rupture; 4) Add 500 μL of Buffer C2, centrifuge for 1 minute, and discard the waste liquid; 5) After washing, open the lid and let it sit for 2-3 minutes; 6) Elute with 60-80 μL of TE Buffer preheated to 60°C, let stand for 5-8 minutes, then centrifuge for 1-2 minutes to collect the DNA.

[0033] The eluted DNA needs to undergo quality testing, including: 1) Concentration detection: The OD260 / 280 ratio should be between 1.7 and 2.0. The detection range is 0.2-100 ng / μL, and the target concentration is ≥10 ng / μL, using the double-stranded DNA quantification method (Qubit HS dsDNA Assay). 2) Electrophoresis detection: 1% agarose gel electrophoresis to observe 16S rRNA gene (about 1.5 kb) or genomic DNA (high molecular weight bands), avoiding degradation (tailing phenomenon); 3) Total microbial count: qPCR quantification: Using the V4 region of the bacterial 16S rRNA gene as the target, qPCR was performed using universal primers (such as 515F / 806R). The copy number of the bacterial 16S rRNA gene in the sample was calculated (≥10^6 copies / μL is acceptable). Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0034] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

[0035] In summary, the above description of the disclosed embodiments enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sample processing method for detecting intestinal flora, characterized in that, include Intestinal flora samples were collected using disposable sterile collection tools and transferred to sterile containers. The collected samples were transported under low temperature conditions or stored at room temperature using stabilizers. The samples were homogenized, impurities were removed, and then aliquoted. Nucleic acid was extracted from gut microbiota samples to obtain complete microbial genomic DNA suitable for high-throughput sequencing.

2. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, The sterile container is a 5mL screw-cap cryovial or an OMNIgene•GUT collection kit. A waterproof label is attached to the outside of the cryovial, indicating the sample ID, collection time, and clinical information.

3. A sample processing method for detecting intestinal flora according to claim 2, characterized in that, The collection of gut microbiota samples specifically involves: For hard stools, use a sterile spoon, wooden scraper or disposable stool collector to collect 2-3g samples from different areas inside the stool; For loose stools, use a disposable sterile spoon or sterile pipette to collect the stool.

4. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, When using the OMNIgene•GUT collection kit, the preservation solution is mixed with feces at a 1:1 volume ratio, vortexed for 10 seconds, and the mixed sample can be stably stored for 14-30 days at room temperature (18-25℃); the preservation solution is selected from any of the following: RNAlater is used to inhibit nuclease activity; 70%-95% ethanol is used to fix the bacterial community structure; PBS buffer containing antibiotics is used to inhibit bacterial growth; When RNAlater is used as the preservation solution, the final concentration should be ≥0.5% (v / v) to effectively inhibit nuclease activity. For short-term preservation (≤1 week), it can be carried out at room temperature. For long-term preservation, it needs to be transferred to 4℃ or -20℃. Processing within 48 hours does not require liquid nitrogen freezing.

5. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, If samples need to be frozen, they should be immediately placed in an environment of -80°C after collection, or briefly stored in an environment of -20°C and then transferred to -80°C. They can also be stored in dry ice for a short period of time.

6. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, The sample homogenization process shall be performed using any of the following methods: Use a bead mill with parameters set to 6.0 m / s for 40-50 seconds. Manual vortex oscillation, time ≥ 2 minutes; Ultrasonic breaker, power 300W, working time 10-15 seconds / cycle, 30-second interval, 3-5 cycles; After homogenization, centrifuge at 3000-5000 rpm for 5 minutes, discard the supernatant, and the precipitate is the bacterial community enrichment.

7. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, The nucleic acid extraction includes the following steps: 1) Take 200 μL of homogenized fecal suspension and add it to the lysis tube; 2) Add 800-1000 μL of PowerBead Solution buffer; 3) Add 80-100 μL of proteinase K, mix thoroughly, and then lyse to release DNA; Nucleic acid extraction from gut microbiota samples should be performed in a biosafety cabinet.

8. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, If the Firmicutes content in the sample is high, an additional 20-30 μL of lysozyme (10 mg / mL) should be added, and the solution should be incubated at 36-39°C for 30 minutes, followed by vortexing for 10-15 minutes until the solution is homogeneous, milky white, and free of visible particles.

9. A sample processing method for detecting intestinal flora according to claim 7, characterized in that, The nucleic acid extraction further includes: 1) After lysis, centrifuge at below 4°C for 1-2 minutes and collect the supernatant; 2) Add 1-2 mL of Buffer C1 and vortex for 5-10 seconds; 3) Add the mixture to the silica gel column in portions, centrifuge for 1-2 minutes, and discard the waste liquid; 4) Add 500 μL of Buffer C2, centrifuge for 1 minute, and discard the waste liquid; 5) After washing, open the lid and let it sit for 2-3 minutes; 6) Elute with 60-80 μL of TE Buffer preheated to 60°C, let stand for 5-8 minutes, then centrifuge for 1-2 minutes to collect the DNA.

10. A sample processing method for detecting intestinal flora according to claim 1, characterized in that, The eluted DNA needs to undergo quality testing, including concentration testing and integrity testing.

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