Method for analyzing and identifying gastric mucosa tissue strains based on 16S rRNA gene sequence

Abstract

The invention discloses a method for analyzing and identifying gastric mucosa tissue strains on the basis of 16S rRNA (ribosomal ribonucleic acid) gene sequences. The identification method comprises the following steps: splitting tissue homogenate and extracting genome DNA (deoxyribonucleic acid); carrying out PCR (Polymerase Chain Reaction) amplification by adopting a 16S rRNA (Ribosomal Ribonucleic Acid) primer, and comparing an amplification product with a gene database to obtain strain classification information of a sample to be detected. The method can be used for rapidly detecting the types of gastric mucosa tissue strains, and the manpower and material resource cost in the microbial species identification process is greatly reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, and in particular relates to a method for identifying bacterial species in gastric mucosal tissue based on 16S rRNA gene sequence analysis. Background Technology

[0002] 16S rRNA is a common ribosomal RNA molecule found in all microorganisms, including bacteria and archaea. Within the cell, 16S rRNA functions as part of the ribosome, participating in protein synthesis and promoter sequence recognition. Its gene sequence is 1500 bp in length. The gene sequence contains highly conserved and variable regions, typically comprising 10 conserved regions (C1-C10) and 9 variable regions (V1-V9). Different bacterial species share the same conserved region sequences; the variable region sequences are used to distinguish and identify different bacterial species.

[0003] Studies show that stomach diseases are increasingly affecting younger people. The microbial community in the healthy human gastric mucosa contains a rich variety of phyla and genera, such as Bacteroidetes, Proteobacteria, Spirulina, Mollusca, and genera like Prevotella, Streptococcus, and Neisseria, playing a crucial role in maintaining the normal physiological structure and function of the gastric mucosa. Helicobacter pylori is a dominant bacterium in the stomach, capable of overcoming the harsh acidic environment of the gastric cavity. Long-term infection can lead to an increase in gastric pH, affecting the health of the gastric mucosa and causing gastric abnormalities such as chronic gastritis, gastric ulcers, and functional dyspepsia.

[0004] Studies have shown that the gastric microbiome is associated with the development and progression of gastric cancer. Helicobacter pylori, by colonizing the gastric mucosa and secreting virulence factors, damages the gastric mucosa, leading to uncontrolled cell proliferation, DNA damage, and disruption of repair mechanisms, ultimately promoting gastric cancer development. The quantity and types of non-Helicobacter pylori bacteria in the stomach also change with disease progression, potentially exerting carcinogenic effects through multiple pathways, including inducing chronic inflammation, oxidative stress, dysregulation of the host immune response, or converting nitrogen compounds in the gastric mucosa into N-nitroso compounds. Identifying structural changes in the gastric mucosa microbiome can provide a basis for early warning of gastric mucosal microbial abnormalities.

[0005] Currently, the most common methods for identifying gastric mucosal bacteria include traditional culture methods, 16S rRNA sequencing, and next-generation sequencing. Traditional microbial identification methods require long culture times and cannot detect pathogens that cannot be cultured under laboratory conditions, limiting a comprehensive understanding of the gastrointestinal flora. High-throughput sequencing technology can provide complete biological information and is suitable for large-scale projects, but due to its complexity and lack of standardized judgment criteria, it may produce erroneous results. 16S rRNA technology amplifies specific regions, accurately identifying bacterial species at a lower cost, with wider applicability, making it more suitable for clinical sample research. Summary of the Invention

[0006] In view of the above-mentioned problems of the prior art, the present invention provides a method for identifying gastric mucosal tissue bacteria based on 16S rRNA gene sequence analysis. The detection method is simple, rapid, highly sensitive and specific.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: 1) The gastric mucosal tissue samples to be tested were collected under aseptic conditions and mixed with lysis buffer, and the tissue homogenate was obtained by physical homogenization; 2) Extract genomic DNA from tissue homogenate; 3) Select 16S rRNA primers to establish a PCR amplification method for bacterial strains; 4) Sequencing the amplified products and performing correlation analysis between the sequencing results and standard sequences to determine the target bacterial species.

[0008] Preferably, the method for preparing the tissue homogenate is as follows: the gastric mucosal tissue sample to be tested is placed in a 1.5 mL centrifuge tube, an appropriate amount of lysis buffer is added and the sample is quickly frozen in a -80°C freezer. After freezing and thawing, the sample is homogenized and then centrifuged. This process is repeated 3 times, and the upper liquid is aspirated to obtain the tissue homogenate.

[0009] Preferably, the mixing ratio of the tissue sample and the lysis buffer is 50~100mg:100~200ul.

[0010] Preferably, the tissue lysis buffer contains 40 mM Tris-HCl, 7 M Urea, 4% CHAPS, 2% NP-40 and 5 mM PMSF.

[0011] Preferably, the physical homogenization is achieved by a homogenizer and a homogenizing rod. The homogenizing rod is rinsed with sterile water, soaked in 75% ethanol solution for 30 minutes, and then disinfected with ultraviolet light for 30 minutes before use.

[0012] Preferably, the 16S rRNA primer sequence is as follows: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' 1492R:5'-TACGGYTACCTTGTTACGACTT-3' FV3: 5'-CCTACGGGNBGCASCAG-3' RV4: 5'-GACTACNVGGGTATCTAATCC-3' Preferably, the correlation analysis involves: splicing the sequencing results using BioEdit analysis software, performing blast sequence alignment using the Genebank database, and identifying the bacterial species based on the sequence similarity level.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The method described in this invention can directly extract nucleic acid DNA from gastric mucosal tissue without the need for in vitro bacterial culture, significantly shortening the identification cycle and reducing costs in the microbial identification process. Using 16S rRNA amplification and sequencing technology expands the detection target from single pathogens to the entire microbial community, greatly enriching the detection range of gastric pathogens. In application, it can help identify gastric-related microbial changes. Furthermore, the gastric mucosal tissue disruption process does not require the use of liquid nitrogen and dry ice, greatly reducing material costs and avoiding operator injury from handling liquid nitrogen and dry ice, making it more environmentally friendly. The use of a homogenizer and homogenizing rod for physical homogenization is simple, flexible, and allows for rapid processing of large numbers of samples, improving experimental efficiency. Attached Figure Description

[0015] Figure 1 The image shows the electrophoresis diagram of PCR amplification of two primer pairs in this embodiment of the invention. The electrophoresis results show that the primer amplification is effective and the bands are single.

[0016] Figure 2 This is the result of comparing the sequencing product sequence of one sample in this embodiment with the Blast database in Genebank. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments.

[0018] Example 1: Extraction of genomic DNA from tissue samples

[0019] 1) Perform this operation in a clean bench. Take 50-100mg of gastric mucosal tissue sample and place it in a 1.5mL centrifuge tube. Add 200ul of lysis buffer (40mM Tris-HCl, 7M Urea, 4% CHAPS, 2% NP-40 and 5mM PMSF) and freeze quickly in a -80℃ freezer. After freezing and thawing, homogenize using a homogenizer and homogenizing stick, and then centrifuge. Repeat the operation 3 times. After that, aspirate the supernatant and transfer it to a new 1.5mL centrifuge tube. The homogenizing stick is rinsed with sterile water, soaked in 75% ethanol solution for 30min, and then sterilized with ultraviolet light for 30min before use.

[0020] 2) Add 20uL Proteinase K and 200uL Buffer AL (QIAGEN), and mix thoroughly by inverting.

[0021] 3) Place at 56℃ for 10 minutes, inverting and mixing several times during this period. The solution will become clear. If the solution does not become completely clear, please extend the lysis time until the solution becomes clear.

[0022] 4) Add 200 μL of anhydrous ethanol, vortex for 15 seconds, and mix thoroughly by inverting.

[0023] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column, place the adsorption column in a collection tube, centrifuge at 8000 rpm for 1 minute, discard the waste liquid, and put the adsorption column back into the collection tube.

[0024] 6) Add 500 μl of washing buffer AW1 (QIAGEN) to the adsorption column, centrifuge at 8000 rpm for 1 minute, discard the waste liquid, and place the adsorption column into the collection tube.

[0025] 7) Add 500 μl of washing buffer AW2 (QIAGEN) to the adsorption column, centrifuge at 14000 rpm for 3 minutes, discard the waste liquid, and place the adsorption column into the collection tube.

[0026] 8) Place the adsorption column back into the collection tube, centrifuge at 14,000 rpm for 1 minute, and discard the waste liquid.

[0027] 9) Transfer the adsorption column to a clean centrifuge tube, and add 50-100 μl of elution buffer AE (QIAGEN) dropwise to the center of the adsorption membrane. Incubate at room temperature for 2-5 minutes, then centrifuge at 8000 rpm for 1 minute. Collect the solution in the centrifuge tube. Determine the DNA concentration and purity, and store at -20℃ for later use.

[0028] Example 2: Detection Experiment Procedure ① Using the nucleic acid DNA extracted in Example 1 as a template, PCR amplification was performed according to the system in Table 1 using the universal bacterial primer 27F / 1492R and the 16S V3-V4 region primer FV3 / RV4 for identifying bacterial diversity.

[0029] Table 1 PCR amplification system Reagent Name Dosage 2*PCR Buffer 10.0 uL dNTPs (2mM) 4.0 uL F (10uM) 0.5 uL R (10uM) 0.5 uL KOD FX (1U / uL) 0.5 uL Template DNA 1.0 uL ddH2O 3.5 uL ②PCR amplification: Place the mixed and centrifuged PCR reaction tube into the PCR instrument and perform amplification according to the procedure in Table 2. Table 2 PCR reaction procedure

[0030] ③ After amplification, perform agarose gel electrophoresis at 120V for 30 minutes and observe using a gel imaging system.

[0031] ④ After recovering the bands, perform sequencing. Take 9 μL of PCR product and add it to 2 μL of the purification system. Follow the procedure in Table 3. purification: Table 3 Purification reaction procedure Reaction phase Reaction conditions purification 37℃ for 50 minutes transsexual 95℃ for 5 minutes save 4℃ ∞ Take 1 μl of the purified product and mix it with the upper and lower sequencing primers according to the system shown in Table 4 below: Table 4 Sequencing reaction system

[0032] Perform the sequencing reaction according to the procedure in Table 5: Table 5 Sequencing reaction procedures

[0033] Precipitation step: Add 2 μl of 125 mmol EDTA to the product of the completed sequencing reaction and let stand for 5 min; add 15 μl of anhydrous ethanol and vortex to mix; centrifuge at 3700 rpm for 30 min; centrifuge upside down for 15 sec, add 50 ml of 70% ethanol and vortex to mix; centrifuge at 3700 rpm for 15 min; centrifuge upside down for 15 sec, place on a 95℃ metal bath; add 10 μl of CBL and denature for 5 min, and finally incubate at -20℃ for 2 min before sequencing.

[0034] ⑤ Analyze the sequencing results using software, compare them with the NT database, identify the types of bacteria present in the sample, and report the results according to the actual situation.

[0035] Example 3 Clinical Sample Testing One clinical gastric mucosal tissue sample was collected. Genomic DNA was extracted, reagents were prepared, PCR amplification and sequencing analysis were performed according to the reagents and methods used in Examples 1 and 2 to verify the feasibility of the method. 1 μL of sample was added to the PCR reaction detection system, and ddH2O was used as the negative control. Electrophoresis results are shown below. Figure 1 As shown, the method used in this invention can be fully applied to the detection of tissue samples, with clear bands and high product yield. The products were subjected to bidirectional sequencing, and the sequencing results were assembled using BioEdit software. Then, BLAST sequence alignment was performed using the Genebank database, and identification was based on sequence similarity levels. Those with a similarity exceeding 99% were identified as the same bacterium. The results (…) Figure 2 The results showed that multiple bacterial species were present in the sample, indicating that this method can quickly and easily identify microorganisms in mucosal tissues.

[0036] The above description is merely a preferred embodiment of the present invention, but the embodiments of the present invention are not limited to the above embodiments. For those skilled in the art, several improvements, modifications, and substitutions can be made without departing from the spirit of the present invention, and these improvements, modifications, and substitutions should also be considered within the protection scope of the present invention.

Claims

1. A method for identifying a bacterial species in a gastric mucosa tissue based on 16S rRNA gene sequence analysis, characterized by, The method comprises the following steps: 1) mixing the collected gastric mucosa tissue sample under sterile conditions with a lysis solution, and obtaining a tissue homogenate by physical homogenization; 2) extracting genomic DNA from the tissue homogenate; 3) selecting 16S rRNA primers and establishing a bacterial PCR amplification method; 4) sequencing the amplification product, and performing correlation analysis on the sequencing result and a standard sequence to determine the bacterial species to be detected.

2. The method of claim 1, wherein, The preparation method of the tissue homogenate in step 1) is as follows: the gastric mucosa tissue sample to be detected is placed in a 1.5 mL centrifuge tube, an appropriate amount of lysis solution is added, and the sample is quickly frozen in a-80℃ refrigerator. After freezing and thawing, the sample is centrifuged, and the operation is repeated for 3 times. Then, the upper liquid is obtained to obtain the tissue homogenate.

3. The method of claim 1, wherein, The mixing ratio of the tissue sample to the lysis solution in step 1) is 50-100 mg: 100-200 ul.

4. The method of claim 1, wherein, The lysis solution in step 1) contains 40 mM Tris-HCl, 7 M urea, 4% CHAPS, 2% NP-40 and 5 mM PMSF.

5. The method of claim 2, wherein, The physical homogenization is achieved by a homogenizer and a homogenizing rod. The homogenizing rod is washed with sterile water, soaked in 75% ethanol solution, and then used after ultraviolet disinfection.

6. The method of claim 1, wherein, The 16S rRNA primer sequence in step 3) is as follows: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' FV3: 5'-CCTACGGGNBGCASCAG-3' RV4: 5'-GACTACNVGGGTATCTAATCC-3'.

7. The method of claim 1, wherein, The correlation analysis in step 4) is as follows: the sequencing result is spliced by using an analysis software, and a Blast sequence comparison is performed by using a Genebank database. The bacterial species is identified according to the sequence similarity level.

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