A method for isolating microorganisms from tissue

Abstract

The present invention discloses a method for isolating microorganisms from tissues. A lysis solution containing the surfactant Triton X100 is used to lyse cells, and the cell membrane of the cells is ruptured by the surfactant Triton X100 without destroying the bacteria containing the cell wall, thereby maintaining the intact structure of the bacteria and being used for subsequent high-throughput sequencing of the metagenome / macrotranscriptome and even single bacterial genome / transcriptome. The method of the present invention can efficiently and accurately isolate microorganisms from tumor tissues, improve the separation efficiency and purity of microorganisms, and avoid the problem of loss of microbial species in existing methods. The method of the present invention is suitable for high-throughput sequencing of microorganisms and can provide high-quality samples and data support for the study of tumor microbiome. The method of the present invention is simple to operate, low-cost, and has broad application prospects and clinical value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating microorganisms from tissues. Background Art

[0002] With the development of bioinformatics and molecular biology, the role of the microbiome in tumorigenesis and progression has received increasing attention. The emergence of high-throughput sequencing technology has provided a powerful tool for in-depth study of tumor-associated microorganisms. However, the number of microorganisms in tumor tissue is relatively small and is contaminated by a large number of host cells and other non-microbial components. Therefore, the efficient isolation of microorganisms from tumor tissue and their high-throughput sequencing remain technical challenges.

[0003] In the prior art, there are several methods for isolating microorganisms from tissues.

[0004] For example, patent application publication number CN115011480A discloses a method for isolating microorganisms from animal tissue and its use. The method involves cutting the tissue into pieces and performing enzymatic hydrolysis. Cycloheximide is then used to remove eukaryotic cells from the animal tissue. The microorganisms are then separated from the animal tissue under varying centrifugal forces. The entire process is complex, and cycloheximide is potentially toxic when used at high concentrations to lyse eukaryotic cells.

[0005] For example, in the method for isolating microorganisms from tissues disclosed in the literature (Bruggeling CE, Garza DR, Achouiti S, Mes W, Dutilh BE, Boleij A. Optimized bacterial DNA isolation method for microbiome analysis of human tissues. Microbiology open. 2021; 10 (3): e1191. doi: 10.1002 / mbo3.1191), proteinase K is used for protein digestion, PBS containing saponin or Triton is used for selective lysis of host cells, and TurboDNAse (DNA enzyme) is used to remove host DNA. However, in this prior art, when DNA enzyme treatment is used, the DNA of the microorganism may also be damaged.

[0006] Therefore, these current methods are often time-consuming, complex, and prone to microbial loss or contamination during operation. Furthermore, existing methods often struggle to avoid interference from host DNA and other impurities during microbial extraction, thus compromising the quality and accuracy of subsequent high-throughput sequencing. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for isolating microorganisms from tumor tissue suitable for high-throughput sequencing of the microbiome. This method can effectively solve the difficulties and problems of isolating microorganisms from tumor tissue in the prior art, improve the extraction efficiency and purity of microorganisms, and provide high-quality samples for subsequent high-throughput sequencing.

[0008] The present invention first provides a lysis solution for isolating microorganisms from tissues, comprising Triton X100 with a final concentration of 0.01wt% to 0.10wt%, 10mM Tris-HCl with a pH of 7.5, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 60U / ml RNase inhibitor, and 0.01wt% BSA.

[0009] The present invention further provides a method for isolating microorganisms from tissue, comprising the following steps:

[0010] (1) Obtain the tissue to be processed, wash the tissue with a pre-cooled buffer containing RNase inhibitors, and then mince the tissue;

[0011] (2) adding the lysis solution of claim 1 for lysis;

[0012] (3) filtering the cleavage product, collecting the filtrate, and centrifuging the filtrate to remove impurities and obtain the supernatant;

[0013] (4) The supernatant obtained in step (3) is centrifuged to separate the bacteria in the tissue.

[0014] Preferably, in step (2), the lysis time is 3 to 15 minutes; after the lysis is completed, a washing buffer is added to terminate the lysis reaction, wherein the washing buffer is 10mM pH 7.5 Tris-HCl, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 60U / ml RI, and 0.01% BSA.

[0015] Preferably, in step (3), when the filtrate is centrifuged to remove impurities, the centrifugal force is 500 g; in step (4), when the supernatant is centrifuged to separate the bacteria in the tissue, the centrifugal force is 3910 g.

[0016] Preferably, the bacteria obtained in step (4) are fixed with formaldehyde; after removing the formaldehyde from the fixed bacteria, they are resuspended in a buffer containing an RNase inhibitor to obtain bacteria isolated from the tissue. When using paraformaldehyde for fixation, the paraformaldehyde weight-to-volume concentration is 4%, and the fixation time is 16 hours. Single-cell transcriptome sequencing of bacteria / microorganisms requires formaldehyde fixation. Metagenomic sequencing does not require formaldehyde fixation. Therefore, whether to add a formaldehyde fixation step can be selected according to the sequencing method and sequencing type.

[0017] Preferably, the tissue to be treated is tumor tissue, colorectal tissue, gastric tissue or other tissue infected by bacteria. In the present application, the tissue to be treated can be any tissue containing microorganisms in cells, the microorganisms are mainly bacteria, and the specific types of bacteria are not limited.

[0018] The present invention also provides a method for high-throughput sequencing of microbial communities in tissues, wherein the method for isolating microorganisms from tissues is used to isolate and extract bacteria from the tissues, and then the isolated and extracted bacteria are sequenced.

[0019] Preferably, the sequencing method is single bacterial transcriptome sequencing, metagenomic sequencing or macrotranscriptome sequencing.

[0020] The cell membrane is ruptured by the surfactant Triton X100 without destroying the bacteria containing the cell wall, thereby maintaining the intact structure of the bacteria for subsequent metagenome / metatranscriptome and even single bacterial genome / transcriptome high-throughput sequencing.

[0021] Beneficial effects of the present invention:

[0022] 1. The method of the present invention can efficiently and accurately separate microorganisms from tumor tissue, improves the separation efficiency and purity of microorganisms, and avoids the problem of loss of microorganism species in existing methods.

[0023] 2. The method of the present invention is suitable for high-throughput sequencing of microbiomes and can provide high-quality samples and data support for the study of tumor microbiomes.

[0024] 3. The method of the present invention is simple to operate, low in cost, and has broad application prospects and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the PI staining result of the bacteria isolated from colorectal cancer tumors in Example 1.

[0026] Figure 2 This is a graph showing the analysis results of the proportion of non-human reads in the sequencing data in Example 1.

[0027] Figure 3 This is a diagram showing the results of single bacterial transcriptome sequencing analysis in Example 1.

[0028] Figure 4 is the percentage of each species in the sequencing data in Example 2.

[0029] Figure 5This is a graph showing the proportion of bacterial data in the total sequencing data in the single bacterial transcriptome sequencing analysis at different lysis times (3 minutes, 5 minutes, 7 minutes, 9 minutes, 11 minutes, 13 minutes, and 15 minutes) when the Triton X100 (mass ratio concentration) concentration is 0.02% in Example 3.

[0030] Figure 6 This is a graph showing the proportion of bacterial data in the total sequencing data in the single bacterial transcriptome sequencing analysis using different Triton X100 (mass ratio concentrations) (0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.10%) when the lysis time is 5 minutes in Example 4. DETAILED DESCRIPTION

[0031] Example 1

[0032] 1. Wash frozen patient-derived colorectal tumor tissue (CRC, sample from Sir Run Run Shaw Hospital affiliated to Zhejiang University, with relevant ethical approval) twice on ice with 2 ml pre-chilled 1× PBS (pH 7.5) + RNase inhibitor (RI, concentration 40 U / ml), and then cut it into small pieces with surgical scissors.

[0033] 2. Immediately add 2 ml of wash buffer (10 mM pH 7.5 Tris-HCl + 146 mM NaCl + 1 mM CaCl2 + 21 mM MgCl2 + 60 U / ml RI + 0.01% BSA (mass ratio concentration)) containing no surfactant (control group 1), 0.05% Tween 20 (mass ratio concentration, control group 2), or 0.05% Triton X100 (mass ratio concentration, experimental group), lyse for 5 minutes, and immediately add 3 volumes of wash buffer to terminate the reaction.

[0034] 3. After the above liquid is filtered through a 40 μm filter membrane, the collected droplets are centrifuged at 500 g / 5 min / 4°C.

[0035] 4. Transfer the supernatant to another centrifuge tube and collect the bacteria in the tissue by horizontal centrifugation at 3910g / 10min / 4°C. Resuspend the bacterial pellet in 1× PBS (pH 7.5) + RI (40U / ml) and centrifuge three times at 800g / 5min / 4°C to discard the pellet until no large fragments that can be stained with propidium iodide (PI) are visible in the supernatant.

[0036] 5. Then, centrifuge at 3910g / 10min / 4°C to collect the bacterial precipitate in the tumor and fix it overnight with 2ml of 4% paraformaldehyde fixative (Biyuntian, P0099-100ml, concentration is mass-to-volume ratio) (perfuse thoroughly to disperse the bacteria as much as possible). At this time, PI staining can be used to examine the number of bacteria and the aggregation. (See the attached results.) Figure 1 Large eukaryotic fragments were found in both control group 1 and control group 2, but not in the experimental group.

[0037] 6. After overnight fixation, remove formaldehyde from the bacterial sample at 3910g / 10min / 4°C, resuspend the precipitate in 1×PBS (pH 7.5) + RI (40U / ml), centrifuge at 800g / 5min / 4°C and discard the precipitate. After passing the supernatant through a 10μm filter, pipette and mix well, and then stain with PI to check the bacterial aggregation and the removal of cell nuclei and other large debris. If large debris still exists, centrifuge again at 800g / 5min / 4°C and discard the precipitate until there are no larger impurities that can be stained with PI.

[0038] 7. Subsequent bacteria were sequenced for single bacterial transcriptome, where the proportion of prokaryotic sequencing data in the overall sequencing data was Figure 2 As shown in the figure, the prokaryotic data accounted for 92% in the experimental group, while the host and other data accounted for 8%, while the prokaryotic data accounted for less than 20% in both control groups 1 and 2. Further dimensionality reduction cluster analysis of bacterial species composition results are shown in the figure. Figure 3 As shown, the individual bacterial species in the experimental group clustered together and had less cross-contamination.

[0039] Example 2

[0040] 1. Wash the frozen colorectal tumor tissue (CRC) twice on ice with 2 ml of pre-cooled 1×PBS (pH 7.5) + RNase inhibitor (RI, 40 U / ml), and then cut it into small pieces with surgical scissors.

[0041] 2. Immediately add 2 ml of 0.05% Triton X100 (mass ratio concentration, experimental group) washing buffer (10 mM pH 7.5 Tris-HCl + 146 mM NaCl + 1 mM CaCl2 + 21 mM MgCl2 + 60 U / ml RI + 0.01% BSA (mass ratio concentration)), lyse for 5 minutes, and immediately add 3 volumes of washing buffer to terminate the reaction.

[0042] 3. After the above liquid is filtered through a 40 μm filter membrane, the collected droplets are centrifuged at 500 g / 5 min / 4°C.

[0043] 4. Transfer the supernatant to another centrifuge tube and collect the bacteria in the tissue by horizontal centrifugation at 3910g / 10min / 4°C. Resuspend the bacterial pellet in 1× PBS (pH 7.5) + RI (40U / ml) and centrifuge three times at 800g / 5min / 4°C to discard the pellet until no large fragments that can be stained with propidium iodide (PI) are visible in the supernatant.

[0044] 5. Then, the bacterial precipitate in the tumor was collected by centrifugation at 3910g / 10min / 4℃, and frozen at -80℃ before being sent to a sequencing company for metagenomic sequencing. The proportion of prokaryotic sequencing data in the entire sequencing data was Figure 4 As shown in the figure, the data measured for the host (human, Homo sapiens) only account for 0.5% of the total sequencing data, and the rest are sequencing data of common intestinal bacteria species.

[0045] Example 3

[0046] 1. Referring to Example 1, the Triton X100 concentration was 0.02%, and the cells were lysed for different times (3 minutes, 5 minutes, 7 minutes, 9 minutes, 11 minutes, 13 minutes, and 15 minutes). The subsequent procedures were performed with reference to Example 1 and single bacterial transcriptome sequencing was performed.

[0047] 2. In the single bacterial transcriptome sequencing results, the proportion of prokaryotic sequencing data in the overall sequencing data is Figure 5 As shown in the figure, the proportion of prokaryotic data at different processing times is above 80%, and all of them have good performance.

[0048] Example 4

[0049] 1. Referring to Example 1, the Triton X100 concentrations (mass ratio) were 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%, respectively. Lysis was performed for 5 minutes. The subsequent procedures were referred to in Example 1 and the single bacterial transcriptome sequencing process was performed.

[0050] 2. In the single bacterial transcriptome sequencing results, the proportion of prokaryotic sequencing data in the overall sequencing data is Figure 6 As shown, in this series of Triton X100 concentrations, the proportion of prokaryotic data is above 80%, and all have good performance.

Claims

1. A method for isolating microorganisms from tissue, characterized in that The following steps are involved: (1) Obtaining the tissue to be processed, washing the tissue with a pre-cooled buffer containing an RNase inhibitor, and then mincing the tissue; the tissue to be processed is colorectal tumor tissue; (2) adding a lysis buffer for lysis; the lysis time is 3 to 15 minutes; the lysis buffer comprises Triton X100 with a final concentration of 0.01 wt% to 0.10 wt%, 10 mM Tris-HCl at pH 7.5, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 60 U / ml RNase inhibitor, and 0.01 wt% BSA; (3) Filter the cleavage product and collect the filtrate, centrifuge the filtrate to remove impurities and obtain the supernatant; (4) centrifuging the supernatant obtained in step (3) to separate the bacteria in the tissue; After lysis was completed, the lysis reaction was terminated by adding a wash buffer consisting of 10 mM Tris-HCl, pH 7.5, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 60 U / ml RI, and 0.01% BSA. In step (3), when the filtrate is centrifuged to remove impurities, the centrifugal force is 500 g; In step (4), when the supernatant is centrifuged to separate the bacteria in the tissue, the centrifugal force is 3910 g.

2. The method for isolating microorganisms from tissue according to claim 1, characterized in that The bacteria obtained in step (4) are fixed with formaldehyde; after removing the formaldehyde from the fixed bacteria, the bacteria are resuspended in a buffer containing an RNase inhibitor to obtain bacteria separated from the tissue.

3. The method for isolating microorganisms from tissue according to claim 2, characterized in that When paraformaldehyde is used for fixation, the paraformaldehyde weight-to-volume concentration is 4%, and the fixation time is 16 hours.

4. A method for high-throughput sequencing of microbial communities in tissues, characterized in that: Bacteria are isolated and extracted from tissue using the method for isolating microorganisms from tissue according to any one of claims 1 to 3, and then the isolated and extracted bacteria are sequenced.

5. The method for high-throughput sequencing of microbial communities in tissues according to claim 4, characterized in that: The sequencing method includes single bacterial transcriptome sequencing, metagenomic sequencing or macrotranscriptome sequencing.

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