Detection method for in-situ labeling of tetrodotoxin in bacteria

Abstract

The invention belongs to the field of bacterial detection of environmental microorganisms, and particularly relates to a detection method for in-situ labeling of tetrodotoxin in bacteria, which comprises the following steps: preparing a slice by using bacteria fixed by a solution containing methanol or ethanol, then carrying out pre-treatment of de-embedding and non-specific sealing, finally carrying out antigen-antibody reaction, and carrying out microscopic examination on a labeling signal by an electron microscope, so as to obtain the tetrodotoxin in-situ labeling detection of the tetrodotoxin in the bacteria. The qualitative and / or quantitative determination of the intracellular tetrodotoxin of the bacteria is realized. The method is high in specificity, good in repeatability and strong in observability, is used for detecting TTX-producing bacteria in the environment, and provides an effective and reliable means for researching the synthesis mechanism of the toxin in the bacteria.

Description

Technical Field

[0001] The invention belongs to the field of bacterial detection of environmental microorganisms, and in particular relates to a detection method for in-situ labeling of tetrodotoxin in bacteria. Background Art

[0002] Tetrodotoxin (TTX) is a highly toxic neurotoxin known for its inhibitory effect on sodium channels. TTX is mainly found in pufferfish and certain other marine organisms. It has attracted much attention due to its high toxicity and potential applications in the medical field, such as analgesia, drug rehabilitation, and anti-arrhythmia. However, the difficulty in obtaining TTX and its low yield limit its application in the medical field. Therefore, the production of TTX by microbial fermentation has become a hot topic of research. Since 1986, researchers have isolated about 150 strains of TTX-producing bacteria from organisms such as pufferfish and blue-ringed octopus. These bacteria mainly belong to the genera Vibrio and Bacillus. These findings provide theoretical support for the microbial origin of TTX and lay the foundation for subsequent research on the production of TTX by microbial fermentation. The detection of TTX is of great significance in the fields of food safety and public health. Ingestion of food containing TTX may lead to serious health problems or even death. In recent years, studies have found that some bacteria can also produce TTX, which provides a new perspective on the source and spread of TTX. Therefore, it is particularly important to develop an accurate and efficient method to detect and quantify bacterial intracellular TTX.

[0003] At present, the detection methods of TTX include bioassay, instrumental analysis, sensor method and immunoassay. These methods are mainly aimed at the detection of animals containing TTX, rather than TTX-producing bacteria. Existing rapid detection products, such as ELISA kits and immunochromatographic test strips, can only detect TTX in animal sample extracts and cannot be used to in situ label bacterial intracellular TTX. In situ labeling of bacterial TTX can directly detect and locate TTX in bacteria, which is of great significance for understanding the biosynthetic pathway of TTX and improving the sensitivity and specificity of detection. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for detecting in situ labeling of tetrodotoxin in bacteria. The method is based on a tetrodotoxin TTX-specific antibody, uses a secondary antibody labeled with nano-gold particles for signal amplification, and establishes an immunohistochemical method for bacterial TTX with high specificity, good repeatability and observability. The method can be used for the detection of TTX-producing bacteria in the environment, and can provide an effective and reliable means for studying the synthesis mechanism of the toxin in bacteria.

[0005] The technical solution of the present invention is a method for detecting in situ labeling of tetrodotoxin in bacteria, comprising the following steps:

[0006] (1) Preparation of sections: The bacteria are first fixed with a solution containing methanol or ethanol, then fixed with a fixative, dehydrated, embedded in gum, and sliced ​​to obtain bacterial sections;

[0007] (2) Pretreatment: remove the embedding gum in the bacterial slices and perform nonspecific blocking;

[0008] (3) Antigen-antibody reaction: first add tetrodotoxin-specific primary antibody and incubate; then add secondary antibody that labels the signal and incubate;

[0009] (4) Detection: Detection of the labeled signal by electron microscopy to conduct qualitative and / or quantitative determination of tetrodotoxin in bacteria.

[0010] In step (1), the solution containing methanol or ethanol includes any one or a combination of pure methanol, pure ethanol, methanol aqueous solution, and ethanol aqueous solution. The methanol aqueous solution is preferably a 90%-99.9% methanol aqueous solution, and the ethanol aqueous solution is preferably a 90%-99.9% ethanol aqueous solution. It is further preferred to fix with pure methanol, pure ethanol, 90%-99.9% methanol aqueous solution, or 90%-99.9% ethanol aqueous solution, and it is further preferred to fix with pure methanol or 90%-99.9% methanol aqueous solution.

[0011] In step (1), the solution containing methanol or ethanol is fixed for 5 minutes, thereby fixing the bacterial structure and increasing its permeability.

[0012] In step (1), the fixative is fixed at 4°C under vacuum for 12 hours, and the fixative is paraformaldehyde / glutaraldehyde (4% / 0.5%) to maintain the bacterial structure. The low temperature of 4°C can prevent the degradation of tetrodotoxin.

[0013] In step (1), gradient dehydration with ethanol is used.

[0014] In step (1), the embedding gum is epoxy resin.

[0015] In step (1), the thickness of the bacterial slice is 50-60 nm.

[0016] In step (1), the bacteria are preferably bacteria that have been subcultured, and are further preferably selected from bacteria that produce tetrodotoxin, including tetrodotoxin-producing bacteria in the genus Vibrio and Bacillus, such as Vibrio alginolyticus and Aeromonas hydrophila. The above-mentioned tetrodotoxin-producing bacteria are further preferably taken from bacteria with the best tetrodotoxin production. The screening method for the best tetrodotoxin production bacteria comprises the following steps: while culturing the bacteria, extracting the tetrodotoxin in the bacterial cells, performing quantitative determination, drawing a tetrodotoxin production-time curve, and screening the cultured bacteria corresponding to the culture time with the best tetrodotoxin production. The quantitative determination method includes an ELISA kit.

[0017] In step (1), the bacteria are washed with a solution containing methanol or ethanol before being fixed, and washed three times with phosphate buffered saline (PBS) to remove impurities.

[0018] In step (2), glycine and sodium periodate are used to remove the embedding gum. The concentration of glycine is 300-800 mM, preferably 500 mM, and the solvent used to prepare glycine is phosphate buffered saline (PBS). The concentration of sodium periodate is 0.008-0.015 g / mL, preferably 0.01 g / mL, and the solvent used to prepare sodium periodate is phosphate buffered saline (PBS).

[0019] In step (2), the non-specific blocking time is 20 minutes, and the blocking agent is bovine serum protein (BSA) and goat serum solution, and the bovine serum protein (BSA) is preferably 5% bovine serum protein (BSA).

[0020] In step (3), before adding the tetrodotoxin-specific primary antibody, the bacterial slices are first washed with phosphate buffer.

[0021] In step (3), the tetrodotoxin-specific primary antibody is incubated at 4°C for 48 h to ensure that the antibody is completely bound to TTX in the bacterial cell.

[0022] In step (3), the concentration of the tetrodotoxin-specific primary antibody is 0.015-0.03 mg / mL, preferably 0.018-0.025 mg / mL, and more preferably 0.02 mg / mL.

[0023] In step (3), after incubation with the tetrodotoxin-specific primary antibody, the sample was washed three times with phosphate buffered saline (PBS), each time for 10 min, to remove excess primary antibody.

[0024] In step (3), the signal-labeled secondary antibody is incubated at room temperature for 2 hours to achieve signal amplification.

[0025] In step (3), after incubation with the secondary antibody that labels the signal, the sample is washed with water to remove excess secondary antibody.

[0026] In step (3), the labeling signal of the secondary antibody includes chromogenic fluorescent molecules, chromogenic metal ions, chromogenic isotopes, etc. For example, the chromogenic metal ions include gold nanoparticles, and the particle size of the gold nanoparticles is 20 nm.

[0027] In step (3), a negative control group is set up, and the negative control group is a primary antibody solution without adding tetrodotoxin antibody.

[0028] In step (4), the bacterial slices are dried before electron microscopy, such as by air drying or air drying.

[0029] In step (4), the method of detecting the marking signal by electron microscopy includes: randomly selecting 9 fields of view to record photos, and counting the positions and quantities of the marking signals.

[0030] In step (4), the qualitative determination of tetrodotoxin in bacteria includes whether tetrodotoxin is produced, the production location of tetrodotoxin, and the distribution location of tetrodotoxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the TTX signal inside the bacterial cell in Example 1. The TTX signal in the positive group is distributed in the cell membrane and cell matrix; the negative control group (primary antibody solution without adding tetrodotoxin antibody) has no positive signal attached.

[0032] Figure 2 This is a schematic diagram of the TTX signal inside the bacterial cell in Example 2. The TTX signal in the positive group is strong and distributed in the cell membrane and cell matrix; the negative control group (primary antibody solution without adding tetrodotoxin antibody) has no positive signal attached.

[0033] Figure 3 Schematic diagram of intracellular TTX signal of Vibrio alginolyticus treated with different concentrations of sodium periodate in Example 3. a: no treatment, b: 0.005 g / mL sodium periodate solution, c: 0.01 g / mL sodium periodate solution, d: 0.02 g / mL sodium periodate solution, e: 0.03 g / mL sodium periodate solution.

[0034] Figure 4 Schematic diagram of TTX signal inside the cell of Vibrio alginolyticus under different cell permeability treatments in Example 4. a: no treatment, b: 100% methanol-5 min, c: 100% ethanol-5 min, d: 1% TritonX100.

[0035] Figure 5 Schematic diagram of intracellular TTX signal of Vibrio alginolyticus treated with different concentrations of TTX antibody in Example 5. The concentration of TTX primary antibody stock solution was 1 mg / mL, a: 20-fold dilution, b: 50-fold dilution, c: 100-fold dilution, d: 200-fold dilution, e: 300-fold dilution, and f: no primary antibody was added. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0037] Example 1

[0038] 1. Preparation of ultrathin sections of Aeromonas hydrophila:

[0039] (1) Inoculate Aeromonas hydrophila into LB medium for recovery. Culture at 28°C for 24 hours to promote bacterial growth. Select a single colony and transfer it to 5 mL of liquid culture medium, and continue to culture for 12 hours to produce TTX. Then collect the bacteria by centrifugation (the mass of the bacteria is ≥1g). Wash the bacteria three times with phosphate buffered saline (PBS) to remove impurities. The bacteria are first fixed with 90% methanol aqueous solution for 5 minutes, and then vacuum fixed with paraformaldehyde / glutaraldehyde solution (4% / 0.5%) for 12 hours to maintain the bacterial structure (should be carried out in a 4°C refrigerator to avoid TTX degradation);

[0040] (2) The samples were dehydrated by ethanol gradient and embedded in epoxy resin to prepare ultrathin sections with a thickness of 50-60 nm. The ultrathin sections were attached to a copper grid to prepare for subsequent testing experiments.

[0041] 2. Immunocolloidal gold in situ labeling of Aeromonas hydrophila TTX:

[0042] (1) Pretreatment

[0043] Take out the copper grid of bacterial ultrathin sections from the 4°C refrigerator and return to room temperature. Use 500mM glycine solution and sodium periodate solution (0.01g / ml) to remove epoxy resin and enhance antigenicity. The solvent for the preparation of glycine solution and sodium periodate solution is phosphate buffered saline (PBS). Use 5% bovine serum protein (BSA) and goat serum solution to block for 20 minutes to reduce nonspecific binding;

[0044] (2) Incubation with primary antibody

[0045] After the copper grid containing the ultrathin section of Aeromonas hydrophila was washed with PBS, 100 μL of TTX-specific primary antibody solution (DPAB-DC4815, Creative Diagnostics) was added to the copper grid. The primary antibody concentration was 0.02 mg / mL and incubated at 4°C for 48 h to ensure that the antibody completely binds to TTX in the bacterial cell. A negative control was set up, and 5% BSA solution was added to the negative control;

[0046] (3) Incubate with secondary antibody labeled with gold nanoparticles

[0047] The copper grid containing the ultrathin sections of Aeromonas hydrophila was washed three times with PBS for at least 10 minutes each time to remove the excess primary antibody. 100 μL of the secondary antibody solution labeled with nanogold particles (20 nm in diameter) (Goat anti-RabbitlgG H&L (20nm Gold) preadsorbed ab27237, abcam) was added to the copper grid and incubated at room temperature for 2 hours to achieve signal amplification. After washing to remove the excess secondary antibody, the sections were dried naturally to prepare for electron microscopy.

[0048] (4) Air-dry and prepare for microscopic examination

[0049] The copper grid containing the ultrathin section of Aeromonas hydrophila was washed in pure water to remove excess secondary antibody, and then naturally dried in preparation for electron microscopy.

[0050] 3. Quantitative analysis of intracellular TTX in Aeromonas hydrophila

[0051] Transmission electron microscopy was used to observe the intracellular TTX labeling signal of Aeromonas hydrophila ( Figure 1 ), set the parameters of TEM Brightfield SA 9300X, High tension: 80kV. Randomly select 9 fields of view to record photos. Count the number of gold nanoparticles, and reflect the production site and quantification of TTX in bacteria based on the location and number of gold nanoparticles.

[0052] Example 2

[0053] 1. Preparation of tissue sections of Vibrio alginolyticus:

[0054] The alginolytic Vibrio strain was inoculated into TCBS medium for recovery. After 24 hours of culture at 37°C, bacterial growth was promoted. A single colony was selected and transferred to 5 mL of liquid culture medium, and the bacterial culture was continued for 12 hours to produce tetrodotoxin. The bacteria were then collected by centrifugation (bacterial mass ≥ 1 g). The bacteria were washed three times with phosphate buffered saline (PBS) to remove impurities. The bacteria were first fixed with 90% methanol aqueous solution for 5 minutes, and then vacuum fixed with paraformaldehyde / glutaraldehyde solution (4% / 0.5%) for 12 hours to maintain the bacterial structure. The sample was dehydrated with ethanol gradient and embedded in epoxy resin to make ultrathin sections with a thickness of 50-60 nm. The ultrathin sections were attached to the copper mesh in preparation for subsequent detection experiments.

[0055] 2. Immunocolloidal gold in situ labeling of Vibrio alginolyticus TTX:

[0056] (1) Pretreatment

[0057] Take out the copper grid of ultrathin sections of Vibrio alginolyticus from the 4°C refrigerator and return to room temperature. Treat the sections with glycine (500mM) and sodium periodate (0.01g / ml) to remove the epoxy resin and enhance antigenicity. Phosphate buffer PBS is used as the solvent for the preparation of glycine solution and sodium periodate solution. Block with 5% bovine serum albumin (BSA) and goat serum solution for 20 minutes to reduce nonspecific binding;

[0058] (2) Incubation with primary antibody

[0059] After the copper mesh containing ultrathin sections of Vibrio alginolyticus was washed with PBS, 100 μL of TTX-specific primary antibody solution was added to the copper mesh, with a primary antibody concentration of 0.02 mg / mL, and incubated at 4°C for 48 h to ensure that the antibody completely binds to TTX in the bacterial cell. A negative control was set up, and a 5% BSA solution was added to the negative control;

[0060] (3) Incubate with secondary antibody labeled with gold nanoparticles

[0061] The copper grid containing ultrathin sections of Vibrio alginolyticus was washed three times with PBS, each time for at least 10 minutes, to remove excess primary antibody. 100 μL of secondary antibody solution labeled with nanogold particles (particle size 20 nm) was added to the copper grid and incubated at room temperature for 2 hours to achieve signal amplification. After washing to remove excess secondary antibody, the sections were dried naturally to prepare for electron microscopy.

[0062] (4) Air-dry and prepare for microscopic examination

[0063] The copper grid containing the ultra-thin sections of Vibrio alginolyticus was washed in pure water to remove excess secondary antibody, and then naturally dried in preparation for electron microscopy.

[0064] 3. Quantitative analysis of intracellular TTX in Vibrio alginolyticus

[0065] Transmission electron microscopy was used to observe the intracellular TTX signal of Vibrio alginolyticus ( Figure 2 ), set the parameters of TEM Brightfield SA 9300X, High tension: 80kV. Randomly select 9 fields of view to record photos. Count the number of gold nanoparticles, and reflect the production site and quantification of TTX in bacteria based on the location and number of gold nanoparticles.

[0066] Example 3

[0067] Comparison of the removal of epoxy resin from the sample surface by sodium periodate at different concentrations. Sodium periodate solutions at different concentrations in Table 1 were used to remove epoxy resin from the surface of Vibrio alginolyticus:

[0068] Table 1 Investigation of treatment with sodium periodate at different concentrations

[0069] Sodium periodate concentration g / 100mL Comparison 0.5 1 2 3 Gold particle density (number of gold particles / um^2) 0 80 240 250 320

[0070] The results are as follows Figure 3 As shown, a: No treatment (0g / mL sodium periodate), epoxy resin on the bacterial surface blocks antibody binding, antibody binding is poor, and the signal is not specific; b: 0.005g / mL sodium periodate treatment for 10 minutes, the bacterial surface structure is intact but resin still exists on the edge; c: 0.01g / mL sodium periodate treatment for 10 minutes, the bacterial surface structure is intact; d: 0.02g / mL sodium periodate treatment for 10 minutes, the bacterial surface structure is corroded and damaged; e: 0.03g / mL sodium periodate treatment for 10 minutes, the bacterial surface structure is severely corroded and damaged. Using 0.01g / mL sodium periodate treatment for 10 minutes is the most appropriate treatment step, which can not only completely remove the epoxy resin on the bacterial surface, but also does not affect the subsequent TTX binding.

[0071] Example 4

[0072] Considering that the slices have a certain thickness, the internal TTX antigen cannot be exposed to affect the antibody binding efficiency, and the cell permeability treatment is increased. The cell permeability treatment solvent is shown in Table 2. The cell permeability treatment solvent is investigated using Vibrio alginolyticus as the object:

[0073] Table 2 Investigation of different cell permeability treatment solvents

[0074]

[0075] The results are as follows Figure 4 As shown, a: No treatment (no cell permeability treatment solvent), less gold particle signal; b: methanol treatment for 10 minutes, gold particle signal increased significantly; c: methanol treatment for 10 minutes, gold particle signal increased significantly, but less than the methanol treatment group signal; d: 1% Triton X 100 treatment group has no significant change, and the number of signals is similar to the control group. Methanol is more suitable as a pre-treatment for TTX antibody binding than ethanol and TritonX100.

[0076] Example 5

[0077] Current studies show that the application of TTX antibodies is mainly concentrated in animal tissues such as pufferfish and goby, while the detection of TTX-producing bacteria has not been reported. In immunohistochemistry detection, the antibody dilution ratio of animal tissue TTX is usually between 1:200 and 1:1000. However, given the variability of TTX content in different samples, too high an antibody concentration may lead to false positive results, thereby misjudging the actual production of TTX in the bacteria; while too low a dilution ratio may result in a signal that is too weak and cannot accurately reflect the true level of TTX in the bacteria. Therefore, the traditional animal antibody dilution ratio is not suitable for the detection of bacterial samples. Based on this, we developed a new optimization strategy to accurately determine the TTX content in TTX-producing bacteria and avoid the problem of false positives or insufficient signals. The concentration of TTX antibodies was investigated using Vibrio alginolyticus as the object, as shown in Table 3:

[0078] Table 3 Treatment with different concentrations of TTX antibodies

[0079]

[0080] The results are as follows Figure 5 As shown, a: The antibody dilution ratio is 1:20. The antibody concentration is too high, and there are a lot of non-specific signals around the bacteria; b: The antibody dilution ratio is 1:50, TTX antibody specifically binds to the bacteria, and there are few non-specific signals around the bacteria; c: The antibody dilution ratio is 1:100, and the overall gold particle signal is significantly reduced; d: The antibody dilution ratio is 1:200, and the overall gold particle signal is significantly reduced; e: 1:300, the overall gold particle signal is significantly reduced; f: Negative control. The antibody dilution ratio of 1:50 for bacteria is the most suitable working concentration.

[0081] When performing bacterial intracellular TTX detection, in order to obtain better detection results, the bacteria are transferred and cultured to ensure the purity and stability of the bacterial strains, while improving the overall enzyme activity of the bacteria to make the bacterial TTX production level more stable. On this basis, the bacteria with the best tetrodotoxin TTX production are screened as the object. The screening method for the best tetrodotoxin production bacteria includes: while culturing the bacteria, extracting the tetrodotoxin in the bacterial cells, using an ELISA kit for quantitative determination, drawing a tetrodotoxin production-time curve, and screening the cultured bacteria corresponding to the culture time with the best tetrodotoxin production.

[0082] It should be pointed out that the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting in situ labeling of tetrodotoxin in bacteria, characterized in that the steps include: (1) Preparation of sections: The bacteria are first fixed with a solution containing methanol or ethanol, then fixed with a fixative, dehydrated, embedded in gum, and sliced ​​to obtain bacterial sections; (2) Pretreatment: remove the embedding gum in the bacterial slices and perform nonspecific blocking; (3) Antigen-antibody reaction: first add tetrodotoxin-specific primary antibody and incubate; then add secondary antibody that labels the signal and incubate; (4) Detection: Detection of the labeled signal by electron microscopy for qualitative and / or quantitative determination of bacterial tetrodotoxin.

2. The detection method according to claim 1, characterized in that: In step (1), the solution containing methanol or ethanol includes any one or a combination of pure methanol, pure ethanol, methanol-water solution, and ethanol-water solution.

3. The detection method according to claim 1, characterized in that: In step (1), the fixative is paraformaldehyde / glutaraldehyde.

4. The detection method according to claim 1, characterized in that: In step (2), glycine and sodium periodate are used to remove the embedding gum.

5. The detection method according to claim 4, characterized in that: The concentration of glycine is 300-800 mM, and the concentration of sodium periodate is 0.008 g / mL-0.015 g / mL.

6. The detection method according to claim 1, characterized in that: In step (2), the non-specific blocking agent is a bovine serum protein and goat serum solution.

7. The detection method according to claim 1, characterized in that: In step (3), the tetrodotoxin-specific primary antibody was incubated at 4°C for 48 h.

8. The detection method according to claim 1, characterized in that: In step (3), the concentration of the tetrodotoxin-specific primary antibody is 0.015-0.03 mg / mL.

9. The detection method according to claim 1, characterized in that: In step (3), the secondary antibody labeled with the signal is incubated at room temperature for 2 hours.

10. The detection method according to claim 1, characterized in that: In step (3), the labeling signal of the secondary antibody includes a chromogenic fluorescent molecule, and / or a chromogenic metal ion, and / or a chromogenic isotope.

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