Method for microfluidic high-throughput screening of AKK bacteria
By using fluorescently labeled antibodies targeting the Amuc-1100 protein of AKK bacteria and microfluidic chip technology, the problem of difficult isolation and purification of AKK bacteria was solved, achieving high-throughput and highly specific screening results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG INST OF TIANJIN UNIV (SHAOXING)
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, the isolation and purification of AKK bacteria is difficult, the screening cycle is long, and the specificity is poor, making it difficult to achieve high-throughput screening.
A fluorescently labeled antibody targeting the Amuc-1100 protein of AKK bacteria was used, combined with microfluidic chip technology, to achieve high-throughput screening of AKK bacteria through droplet generation and culture.
High-throughput screening of AKK bacteria was achieved with a simple and highly specific process, improving screening efficiency.
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Figure CN122011176A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and specifically relates to a microfluidic high-throughput screening method for AKK bacteria (Akkermansia muciniphila, AKK). Background Technology
[0002] Akkermansia muciniphila (A. muciniphila, referred to as AKK bacteria in this article) belongs to the phylum Verrucous microbes and is a Gram-negative bacterium widely distributed in vertebrates. It mainly colonizes the mucus layer of the cecum and colon, accounting for 1%-3% of the total intestinal microbiota. AKK bacteria grow in the intestine by utilizing mucin secreted by the host as "food," thereby establishing themselves in the intestine through competitive exclusion and protecting the intestine from pathogens.
[0003] Recent studies have shown that AKK bacteria exhibit beneficial effects on various pathologies, with a wide range of discovered pharmacological effects. It has been widely reported to possess multiple efficacy pathways, including anti-cancer, anti-aging, lipid-lowering and anti-inflammatory effects, immune enhancement, and regulation of the nervous system. Studies have shown that oral administration of 1×10⁻⁶ spores to human volunteers... 10 Both live and dead AKK bacteria are very safe in terms of dosage, with no adverse reactions reported. This makes AKK bacteria one of the most promising next-generation probiotics.
[0004] Although *Acanthopanax* (AKK) bacteria have various potential applications, their isolation and purification as a strict anaerobic bacterium is extremely difficult. Since *Acanthopanax* was first isolated from human feces by Derrien et al. in 2004, plating screening has been the mainstream screening method. However, plating screening for *Acanthopanax* is time-consuming and lacks specificity, posing a challenge to rapid screening of *Acanthopanax*. Finding new high-throughput screening methods for *Acanthopanax* is of great significance for rapid and accurate screening. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fluorescently labeled antibody against the Amuc-1100 protein of AKK bacteria.
[0006] The second objective of this invention is to provide a microfluidic high-throughput screening method for AKK bacteria.
[0007] The technical solution of this invention is summarized as follows: A fluorescently labeled antibody against the Amuc-1100 protein of *AKK* bacteria was prepared using the following steps: An antibody against the Amuc-1100 protein of *AKK* bacteria was prepared by adding PBS buffer to form an antibody solution. fluorescein isothiocyanate was added to dimethyl sulfoxide (DMSO) to prepare a fluorescein isothiocyanate-DMSO solution. The fluorescein isothiocyanate-DMSO solution was then added to the antibody solution, vortexed, incubated, and eluted according to the antibody labeling kit instructions. The resulting purified antibody was a fluorescently labeled antibody against the Amuc-1100 protein of *AKK* bacteria, referred to simply as the fluorescently labeled antibody. The antibody against the Amuc-1100 protein of AKK bacteria comprises a heavy chain variable region and a light chain variable region; HCDR1 in the heavy chain variable region The amino acid sequences of 3 are as shown in SEQ ID NO:1 As shown in Figure 3; LCDR1 in the light chain variable region The amino acid sequences of 3 are as shown in SEQ ID NO:9 As shown in Figure 11.
[0008] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4.
[0009] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:12.
[0010] A microfluidic high-throughput screening method for AKK bacteria includes the following steps: 1) Prepare a microfluidic chip, which includes a U-shaped droplet generation channel (3). The middle of the left channel of the U-shaped droplet generation channel (3) is connected to the mobile phase injection cell (1); the middle of the right channel of the U-shaped droplet generation channel (3) is connected to the dispersed phase injection cell (2); the middle of the right channel of the U-shaped droplet generation channel (3) is connected to the S-shaped droplet flow channel (4) through a channel and then connected to the droplet culture cell (5) through a channel; the droplet culture cell (5) is connected to the droplet collection cell (6) through a channel. 2) Fresh feces were collected from healthy volunteers and subjected to 10 minutes of sterile saline solution in an anaerobic incubator. -3 -10 -5 The diluent was obtained by serial dilution, and after dilution with modified brain and heart infusion broth medium, fluorescently labeled antibody according to any one of claims 1-3 was added to obtain the dispersed phase; 3) Under anaerobic culture conditions, a mixture of paraffin oil and Span-80 was used as the mobile phase and introduced into the mobile phase injection cell (1) of the microfluidic chip; the dispersed phase was introduced into the dispersed phase injection cell (2) of the microfluidic chip to prepare droplets. The generated droplets entered the droplet culture cell (5) through the S-shaped droplet flow channel (4). When the droplet culture cell (5) was filled with droplets, the introduction of the dispersed phase and the mobile phase was stopped. The culture was carried out at a temperature of 35℃-40℃ for 24-48 hours. After the culture was completed, the mobile phase was introduced again so that the droplets in the droplet culture cell (5) could be introduced into the droplet collection cell (6). The droplets were taken out and subjected to fluorescence microscopy. The fluorescence signal was detected by the fluorescence microscope receiver. The droplets with fluorescence signals were cultured on the modified brain heart infusion broth solid medium for 24-48 hours to obtain the strain to be identified. 4) Using the strain to be identified as a template, PCR amplification was performed using upstream primer P1 and downstream primer P2 as upstream and downstream primers, the amplification products were sequenced, and the sequencing results were compared with BLAST sequences on the NCBI website to screen out the target strain AKK. The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO.17; The nucleotide sequence of the downstream primer P2 is shown in SEQ ID NO.18.
[0011] Advantages of this invention: The microfluidic high-throughput screening method for AKK bacteria of the present invention has the characteristics of high throughput, simple experimental procedure and high specificity, which can improve the screening efficiency of AKK bacteria. Attached Figure Description
[0012] Figure 1 This is a schematic diagram of a microfluidic chip structure. Detailed Implementation
[0013] The antibody labeling kit used in this invention (commercial product, model: Thermo Fisher Zip Alexa Fluor Antibody Labeling Kit).
[0014] The present invention will be further described below through specific embodiments.
[0015] Example 1 Recombinant expression of Amuc-1100 protein 1.1 PCR amplification of the Amuc-1100 gene Akkermansia muciniphila ATCC BAA was extracted using a bacterial genomic DNA extraction kit (Tiangen, China). The genome of *Akkermansia muciniphila* ATCC BAA-835 (purchased from the China General Microbiological Culture Collection Center) was used as a reference for the sequence in Amuc-RS05900 hypothprotein [Akkermansia muciniphila ATCC BAA-835] (Gene ID: 34174504) on NCBI. Primers P3 (SEQ ID NO. 19) and P4 (SEQ ID NO. 20) were designed for homologous recombination (SEQ ID NO. 19: 5'-GTGGTGGTGGTGGTGCTCGAGGGTACCATATGATCGTCAAT-3'; SEQ ID NO. 20: 5'-TAAGAAGGAGATATACATATGCCTTGGCTCGAGATCTTCAG-3'). Primers P3 and P4 were synthesized by Beijing Qingke Biotechnology Co., Ltd. The extracted *Akkermansia muciniphila* genome was amplified by PCR in a 25 μL system (12.5 μL Taq 2×Master PCR). Mixture (Takara, China), 2 μL template DNA (Akkermansia myxophilus genome), 1 μL each of forward and reverse primers P3 and P4, and 9.5 μL ddH2O were used in a PCR instrument (Bio-Rad, USA). The reaction program was: 95℃ pre-deformation for 3 min, 95℃ for 30 s, 72℃ for 1 min, for a total of 30 cycles, and a final extension at 72℃ for 15 min to obtain the amplified product. The amplified product was recovered according to the DNA gel extraction kit (Beyotime) instructions to obtain the Amuc-1100 gene.
[0016] 1.2 Construction of recombinant expression plasmids The Amuc-1100 gene and vector pET-26b (commercial) were mixed at a molar ratio of 6:1 and ligated with T4 DNA ligase at 23°C for 1 hour to obtain the pET-26b-Amuc-1100-vector. The pET-26b-Amuc-1100-vector was transformed into *E. coli* competent cells DH5α according to the instructions of the competent cell preparation and transformation kit (Beyotime). The transformed DH5α cells were inoculated into LB solid medium containing 50 μg / mL ampicillin and cultured at 37°C for 12 hours. Single colonies were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C for 12 hours. Plasmid DNA was extracted according to the plasmid miniprep kit (Beyotime) instructions and subjected to NdeI and XhoI double enzyme assays to obtain the pET-26b-Amuc-1100 plasmid.
[0017] 1.3 Induction and purification of Amuc-1100 gene Following the instructions of the competent cell preparation and transformation kit (Beyotime), the pET-26b-Amuc-1100 plasmid was transformed into *E. coli* strain BL21 to obtain strain BL21-pET-26b-Amuc-1100. The BL21-pET-26b-Amuc-1100 strain was inoculated into 5 mL of LB broth and cultured at 37°C for 12 h. After 12 h, the bacterial culture was inoculated into LB broth at a volume ratio of 1:100 and cultured at 37°C until OD500 was reached. 600 The concentration was 0.7 ± 0.1. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mmol / L to induce Amuc-1100 protein expression. The culture was continued at 37℃ for 6 h to obtain the BL21-pET-26b-Amuc-1100 fermentation broth. The BL21-pET-26b-Amuc-1100 fermentation broth was placed in an ice bath and sonicated for 15 min to disrupt the cells. The cells were then centrifuged at 10000 r / min for 15 min at 4℃ to obtain a supernatant containing Amuc-1100 protein. The supernatant was purified using nickel ion chelate aminotriacetic acid affinity chromatography. The filtrate at a molecular weight of 70 kDa was collected to obtain an aqueous solution of Amuc-1100 protein. The concentration of Amuc-1100 protein was determined using the Bradford method (Coomassie Brilliant Blue assay).
[0018] Example 2: Preparation of Amuc-1100 protein antibody AMUC-1 2.1 Animal Immunization The Amuc-1100 protein obtained in Example 1.3 was emulsified with complete Freund's adjuvant and immunized 6-8 week old BALB / c mice subcutaneously at a dose of 50 μg / mouse. A second immunization was performed two weeks later with Amuc-1100 protein emulsified with incomplete Freund's adjuvant at a dose of 50 μg / mouse. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. Mice with the highest antibody titers were selected for cell fusion.
[0019] 2.2 Cell Fusion SP2 / 0 myeloma cells derived from BALB / c were cultured in complete culture medium at 37°C and 5% CO2 for 15-20 hours until the logarithmic growth phase. Following the instructions of the cell fusion kit (Shanghai Yuanye), spleens from mice immunized in step 2.1 were used to prepare a lymphocyte suspension. The lymphocyte suspension was mixed with the myeloma cells at a ratio of 1:5. 1 mL of 50% PEG aqueous solution (pH=8.0) at 37°C was added, along with incomplete culture medium and its stop solution. After centrifugation and discarding the supernatant, HAT culture medium was added to suspend the cells, and the mixture was brought to a final volume of 50 mL. The cells were then aliquoted into 3.5 cm culture dishes and incubated at 37°C and 5% CO2.
[0020] 2.3 Screening and Cloning Cell clones were selected 7-10 days after fusion and ELISA was performed using Amuc-1100 protein according to the ELISA kit instructions (Thermo Fisher Scientific). Positive cell lines were labeled. Positive cells were diluted with HAT medium to a cell concentration of 50-60 cells / mL. ELISA values were measured 5-6 days after each dilution until a positive result was obtained for the entire 96-well plate, yielding a monoclonal cell suspension.
[0021] 2.4 Preparation and purification of antibody against Amuc-1100 protein of AKK bacteria (AMUC-1 for short) Male BALB / c mice aged 10-12 weeks were injected intraperitoneally with 0.5 mL of terbutaline. One week later, each mouse was injected intraperitoneally with a 1 mL syringe containing a suspension of monoclonal cells washed and resuspended in PBS. The cell volume was 5 × 10⁶ cells. 6 Each mouse was tested. After ascites fluid accumulated, it was collected, centrifuged at 2000 rpm for 15 min, and the supernatant was collected. The ascites fluid was purified by protein A column chromatography. AMUC-1 was obtained after purification and sent for sequencing. The sequence information is shown in Table 1.
[0022] Table 1
[0023] Example 3: Fluorescent labeling of AMUC-1 antibody 3.1 Preparation of antibody solution Take 0.5 mL of AMUC-1 antibody, add 10 mM phosphate buffer to adjust the antibody concentration to 2 mg / mL, and adjust the pH of the solution to 9.0 (pH 9.0-9.5 is acceptable) using 10% sodium bicarbonate aqueous solution to obtain the antibody solution.
[0024] 3.2 Preparation of fluorescein solution Under light-protected conditions, 0.5 mg of fluorescein isothiocyanate was added to 500 μL of dimethyl sulfoxide and vortexed to mix. The concentration of fluorescein isothiocyanate was adjusted to 10 mg / mL to obtain a fluorescein isothiocyanate-dimethyl sulfoxide solution.
[0025] 3.3 Preparation of fluorescently labeled antibodies Under light-protected conditions, the fluorescein isothiocyanate-dimethyl sulfoxide solution prepared in step 3.2 was added to the antibody solution in step 3.1. The mixture was shaken and incubated at 35°C for 40 min to obtain the labeled product. Following the instructions of the antibody labeling kit (Thermo Fisher Scientific), the labeled product after incubation was added to an EP tube equipped with a centrifuge column. The tube was incubated at room temperature for 3 min, centrifuged at 800 rpm for 2 min, and the supernatant was discarded. The labeled product on the centrifuged column was dissolved in 5 volumes of 10 mM phosphate buffer to obtain a fluorescently labeled antibody against the Amuc-1100 protein of AKK bacteria, referred to as the fluorescently labeled antibody, which was stored at 4°C in the dark.
[0026] Example 4: Fabrication of a Microfluidic System 4.1 Fabrication of Microfluidic Chip Templates Design a microfluidic chip mask with the same structure as the microfluidic chip using AutoCAD software.
[0027] After ultrasonically cleaning the silicon wafer by sequentially immersing it in acetone, methanol, and isopropanol for 10-15 minutes, it is dried with nitrogen. A layer of SU-8 photoresist is then spread on the cleaned silicon wafer. A designed microfluidic chip mask is added on top of the photoresist. After UV exposure, the wafer is placed in a developing solution. The SU-8 photoresist covered by the microfluidic chip mask is cured, while other areas are dissolved, thus obtaining the microfluidic chip template.
[0028] 4.2 Preparation of Microfluidic Chip Semi-finished Products Weigh polydimethylsiloxane (PDMS) and curing agent (Dow Corning SYLGARD 184) in a mass ratio of 10:1, and stir thoroughly to obtain a mixed adhesive. Pour the mixed adhesive into the microfluidic chip template prepared in 4.1, place it in a vacuum chamber and evacuate for 5 hours to eliminate all air bubbles in the mixed adhesive. Place it in an oven and bake at 80°C for 3 hours to cure the PDMS and obtain a crude microfluidic chip. Use a punch with an outer diameter of 1.4-1.6 mm to punch holes in the mobile phase injection cell, dispersed phase injection cell, and droplet collection cell of the crude microfluidic chip to obtain a semi-finished microfluidic chip.
[0029] 4.3 Fabrication of Microfluidic Chips A 25×75mm glass slide was sequentially immersed in acetone, methanol, and isopropanol, each ultrasonically cleaned for 10-15 minutes, and then dried with nitrogen. The microfluidic chip semi-finished product and the glass slide were then surface-treated using an oxygen plasma surface treatment instrument. The oxygen vacuum was set to 0.3-0.5 mbar, and the treatment time was 50 seconds. The treated microfluidic chip semi-finished product was then tightly bonded to the glass slide and placed in an oven at 80℃ for 8-10 hours. After treatment, a 1.6-1.7mm PE tube was inserted into the perforated holes in the mobile phase injection cell, dispersed phase injection cell, and droplet collection cell to obtain the microfluidic chip.
[0030] The microfluidic chip includes a U-shaped droplet generation channel 3. The middle of the left channel of the U-shaped droplet generation channel 3 is connected to the mobile phase injection cell 1; the middle of the right channel of the U-shaped droplet generation channel 3 is connected to the dispersed phase injection cell 2; the middle of the right channel of the U-shaped droplet generation channel 3 is connected to the S-shaped droplet flow channel 4 through a channel, and then connected to the droplet culture cell 5 through a channel; the droplet culture cell 5 is connected to the droplet collection cell 6 through a channel. Example 5: A microfluidic high-throughput screening method for AKK bacteria, comprising the following steps: 5.1 Preparation of the dispersed phase Add 1g of fresh feces collected from healthy volunteers to a sterile centrifuge tube. Then, in an anaerobic incubator (the system of the anaerobic incubator consists of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide by volume), perform a serial dilution with sterile physiological saline to a concentration of 10:1. -3 -10 -5 The diluent was obtained by gradient dilution. The diluent was diluted tenfold with modified brain and heart infusion broth medium (brain and heart infusion broth medium + 0.4% wt mucin). The fluorescently labeled antibody (prepared in Example 3) was then added to make the final concentration of the fluorescently labeled antibody 2-5 ug / mL to obtain the dispersed phase.
[0031] 5.2 Microdroplet Preparation and Cultivation Based on Microfluidic Chips Under anaerobic culture conditions (the anaerobic culture system consists of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide by volume), paraffin oil + Span-80 (paraffin oil:Span-80 volume ratio of 94%:6%) was used as the mobile phase at a flow rate of 1-1.5 μL / min and introduced into the mobile phase injection cell 1 of the microfluidic chip; the dispersed phase prepared in step 5.1 was introduced into the dispersed phase injection cell 2 of the microfluidic chip at a flow rate of 0.5-1 μL / min for droplet preparation. The generated droplets entered the droplet culture cell 5 through the S-shaped droplet flow channel 4. After 10 min of droplet preparation, when the droplet culture cell 5 was filled with droplets (approximately 5000-8000 droplets), the droplets were cultured. After that, stop the introduction of the dispersed phase and the mobile phase, and incubate at 37℃ (35℃-40℃ is acceptable) for 24 hours (24-48 hours is acceptable). After the incubation is completed, reintroduce the mobile phase at a flow rate of 0.5-1uL / min, so that the droplets in the droplet culture tank 5 are introduced into the droplet collection tank 6. Take them out and perform fluorescence microscopy detection. The fluorescence detection wavelength is 550nm. Detect the fluorescence signal through the fluorescence microscope receiver. Collect the droplets corresponding to the fluorescence signal >10000AU and incubate them on modified brain heart infusion broth solid medium (brain heart infusion broth medium + 0.4%wt mucin + 1.5%wt agar powder) for 24 h (24-48 h is acceptable) to obtain the strain to be identified.
[0032] 5.3 Identification of AKK strains Using the bacterial strain to be identified collected and cultured in section 5.2 as a template, and upstream primer P1 and downstream primer P2 as upstream and downstream primers, PCR amplification was performed. The reaction was performed in a 25 μL system (12.5 μL Taq Plus Master Mix II, 2 μL of bacterial culture to be identified, 1 μL each of forward and reverse primers P1 (SEQ ID NO.17) and P2 (SEQ ID NO.18), and 9.5 μL ddH2O) on a PCR instrument (Bio-Rad, USA). The reaction program was as follows: 94℃ pre-deformation for 3 min, 94℃ denaturation for 30 s, 54℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles, and a final extension at 72℃ for 15 min to obtain the amplified product. The amplified product was sent to Genewiz Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with BLAST sequences on the NCBI website. Ten random sequencing comparison results are shown in Table 2. The results show that the microfluidic chip combined with fluorescently labeled antibody screening of AKK bacteria has extremely high accuracy and can improve the screening efficiency of AKK bacteria.
[0033] SEQ ID NO. 17: 5'-CAGCACGTGAAGGTGGGGAC-3'.
[0034] SEQ ID NO. 18: 5'-ATCTGAAGCCAACCGCAAGG-3'.
[0035] Table 2 shows the results of high-throughput sorting of AKK bacteria using microfluidic chips combined with fluorescently labeled antibodies.
Claims
1. A fluorescently labeled antibody against the Amuc-1100 protein of *AKK* bacteria, characterized in that... It is prepared using the following steps: An antibody against the Amuc-1100 protein of *AKK* bacteria was prepared by adding PBS buffer to form an antibody solution. fluorescein isothiocyanate was added to dimethyl sulfoxide (DMSO) to prepare a fluorescein isothiocyanate-DMSO solution. The fluorescein isothiocyanate-DMSO solution was then added to the antibody solution, vortexed, incubated, and eluted according to the antibody labeling kit instructions. The resulting purified antibody was a fluorescently labeled antibody against the Amuc-1100 protein of *AKK* bacteria, referred to simply as the fluorescently labeled antibody. The antibody against the Amuc-1100 protein of AKK bacteria comprises a heavy chain variable region and a light chain variable region; HCDR1 in the heavy chain variable region The amino acid sequences of 3 are as shown in SEQ ID NO:1 As shown in Figure 3; LCDR1 in the light chain variable region The amino acid sequences of 3 are as shown in SEQ ID NO:9 As shown in Figure 11.
2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:
4.
3. The antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:
12.
4. A microfluidic high-throughput screening method for AKK bacteria, characterized in that... Includes the following steps: 1) Prepare a microfluidic chip, which includes a U-shaped droplet generation channel (3). The middle of the left channel of the U-shaped droplet generation channel (3) is connected to the mobile phase injection cell (1); the middle of the right channel of the U-shaped droplet generation channel (3) is connected to the dispersed phase injection cell (2); the middle of the right channel of the U-shaped droplet generation channel (3) is connected to the S-shaped droplet flow channel (4) through a channel and then connected to the droplet culture cell (5) through a channel; the droplet culture cell (5) is connected to the droplet collection cell (6) through a channel. 2) Fresh feces were collected from healthy volunteers and subjected to 10 minutes of sterile saline solution in an anaerobic incubator. -3 -10 -5 The diluent was obtained by serial dilution, and after dilution with modified brain and heart infusion broth medium, fluorescently labeled antibody according to any one of claims 1-3 was added to obtain the dispersed phase; 3) Under anaerobic culture conditions, a mixture of paraffin oil and Span-80 was used as the mobile phase and introduced into the mobile phase injection cell (1) of the microfluidic chip; the dispersed phase was introduced into the dispersed phase injection cell (2) of the microfluidic chip to prepare droplets. The generated droplets entered the droplet culture cell (5) through the S-shaped droplet flow channel (4). When the droplet culture cell (5) was filled with droplets, the introduction of the dispersed phase and the mobile phase was stopped. The culture was carried out at a temperature of 35℃-40℃ for 24-48 hours. After the culture was completed, the mobile phase was introduced again so that the droplets in the droplet culture cell (5) could be introduced into the droplet collection cell (6). The droplets were taken out and subjected to fluorescence microscopy. The fluorescence signal was detected by the fluorescence microscope receiver. The droplets with fluorescence signals were cultured on the modified brain heart infusion broth solid medium for 24-48 hours to obtain the strain to be identified. 4) Using the strain to be identified as a template, PCR amplification was performed using upstream primer P1 and downstream primer P2 as upstream and downstream primers, the amplification products were sequenced, and the sequencing results were compared with BLAST sequences on the NCBI website to screen out the target strain AKK. The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO.17; The nucleotide sequence of the downstream primer P2 is shown in SEQ ID NO.18.