Method for simultaneously detecting bioavailability of adsorbed and desorbed antibiotics in soil

Abstract

The invention discloses a method for simultaneously detecting bioavailability of adsorbed and desorbed antibiotics in soil, and belongs to the field of biotechnology application. A biosensing detection system is designed by adopting a method of combining fluorescence sensing bacteria and an agar plate, and the biosensing detection system is used for detecting the biosensing bacteria by controlling the humidity of an agar plate system based on soil-antibiotic interface behaviors and an agar diffusion principle. The system can detect and quantify the bioavailability of tetracycline adsorbed and desorbed in soil with different water potentials. The biosensing detection system designed by the invention is simple, sensitive, low in consumption and convenient, soil with the water potential range of-10.59 to-2.95 Kpa can be detected in situ, and the lowest detection limit value of tetracycline antibiotics is 1mg / kg.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil. Background Art

[0002] The overuse of antibiotics in human medicine and animal agriculture has led to the occurrence and development of antibiotic resistance in the environment. Antibiotic resistance genes (ARGs) can be horizontally transferred between bacteria. Pathogens that acquire ARGs can weaken the effect of clinical antibiotic treatment, which undoubtedly poses a serious threat to human health. Tetracycline antibiotics are one of the most commonly used drugs in animal production. Most of them are excreted through animal excrement, and then through manure and sewage reuse, resulting in widespread tetracycline residues in the soil. The presence of tetracycline in soil is a dynamic process, usually adsorbed by soil particles (including minerals and organic matter, etc.) and subsequently desorbed. Understanding the acquisition and absorption of adsorbed and desorbed tetracycline antibiotics in soil by bacteria (i.e., bioavailability) is crucial to affecting the generation and spread of ARGs in soil microbial communities. However, there is currently no effective means for the simultaneous detection of adsorbed and desorbed antibiotics in the world. Therefore, it is urgent to develop technical methods that can simultaneously detect the bioavailability of adsorbed and desorbed tetracycline in soil and apply them to actual environmental testing.

[0003] At present, the use of fluorescent sensing bacteria to detect antibiotics or other organic compounds has been recognized, and it is a feasible technical means to use it to detect the bioavailability of antibiotics in the environment. However, the current inventions or research are mainly aimed at the development and construction of fluorescent sensing bacteria, and there are few inventions on how to apply these strains to detect the bioavailability of antibiotics in the actual environment, especially their application in the simultaneous detection of the bioavailability of adsorbed and desorbed antibiotics in soil. This mainly involves the stability, detection limit and long-term detection ability of fluorescent sensing bacteria under complex environmental conditions. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil.

[0005] The technical solution adopted by the present invention is: a biosensor detection system, including a solid culture medium containing agar containing screening antibiotics, and E.coli pTGM fluorescent sensing bacteria coated on the solid culture medium containing agar, and the E.coli pTGM fluorescent sensing bacteria can respond to the screening antibiotics.

[0006] Preferably, the E. coli pTGM fluorescent sensor bacteria contains a pTGM plasmid carrying a tetracycline resistance gene tet (M), a transcriptional regulatory gene tetR and a green fluorescent protein gene (gfp).

[0007] Preferably, the culture medium is LB solid medium containing 100 mg / L ampicillin and 2% (w / v) agar.

[0008] Preferably, E. coli pTGM fluorescent sensing bacteria are cultured to an OD of 600 When the concentration is 0.4-0.6, apply it on the culture medium.

[0009] The method for detecting the bioavailability of antibiotics in soil using a biosensor detection system is as follows: a soil sample to be tested is placed on a solid culture medium, and after incubation, fluorescent substances are observed around the soil sample to determine whether the soil sample contains bioavailable antibiotics.

[0010] Preferably, the antibiotic is a tetracycline antibiotic, preferably one or more of tetracycline, oxytetracycline and chlortetracycline;

[0011] Simultaneously detect adsorbed bioavailable antibiotics and / or desorbed bioavailable antibiotics in soil.

[0012] Preferably, the specific method is as follows:

[0013] Step 1: Take the unknown soil to be tested, grind, sieve and sterilize it in sequence;

[0014] Step 2: Add 2% (w / v) agar to LB medium, add ampicillin to 100 mg / L after sterilization, and place in a culture dish to solidify; spread E. coli pTGM fluorescent sensor bacteria containing pTGM plasmid carrying tetracycline resistance gene tet (M), transcriptional regulatory gene tetR and green fluorescent protein gene (gfp) on the culture dish;

[0015] Step 3: Place the soil sample on the culture medium and observe whether fluorescent apertures are produced after incubation to determine whether the soil sample contains bioavailable antibiotics.

[0016] Preferably, the method further comprises step 4:

[0017] Step 4: Collect the bacterial flora on the soil sample and the aperture range culture medium sample respectively for extraction, detect and calculate the average fluorescence intensity, and obtain the concentration of bioavailable antibiotics in the sample based on the standard curve.

[0018] Preferably, the soil and agar surfaces can be imaged directly using a laser confocal microscope to detect the fluorescence intensity of the formed fluorescent substance;

[0019] Alternatively, E. coli pTGM colonies were extracted using iotrolan density gradient separation and the fluorescence intensity of each cell was measured using flow cytometry.

[0020] Preferably, the water potential range of the soil sample to be tested is -10.59 to -2.95 KPa.

[0021] The advantages and positive effects of the present invention are as follows: the biosensor detection system involved in the present invention is convenient, fast, highly sensitive, low-consumption and environmentally friendly, and can realize the simultaneous detection of the bioavailability of tetracycline antibiotics in adsorbed and desorbed states in soil; and the biosensor detection system has a wide range of uses and can be applied to soils with a variety of different water potentials, and can simultaneously detect the bioavailability of antibiotics in adsorbed and desorbed states therein, providing important technical support for timely and effective monitoring of the behavior of antibiotics in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the detection results of the method of the present invention, wherein (a), (b) and (c) correspond to clay loam, sandy clay loam and loamy sand, respectively;

[0023] Figure 2 The relationship between fluorescence intensity and bioavailable tetracycline concentration; A is bacteria isolated from the soil surface; B is bacteria isolated from the agar surface 0.75 cm outside the soil;

[0024] Figure 3 is a graph showing the relationship between the diameter and intensity of the fluorescent ring and the bioavailable tetracycline in the method of the present invention;

[0025] Figure 4 This is a graph showing the relationship between the fluorescence intensity of the fluorescent sensing bacteria and the soil water potential in the method of the present invention. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0027] The present invention relates to a method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil. A biosensing detection system is designed by combining fluorescent sensing bacteria with agar plates. Based on the soil-antibiotic interface behavior and agar diffusion principle, the fluorescent sensing bacteria can form bright green fluorescent bacteria around the surface of the culture medium (containing desorbed tetracycline) and / or the surface of the soil (containing adsorbed tetracycline), which proves that the sample object detected contains antibiotic bioavailability. Tetracycline antibiotics can be one or more of tetracycline, oxytetracycline and chlortetracycline.

[0028] Considering the wide range of temporal and spatial characteristics of moisture differences in natural surface soil, by adjusting the humidity of the agar plate system, the system can also detect and quantify the bioavailability of tetracycline antibiotics in adsorbed and desorbed states in soils with different water potentials. The biosensor detection system designed by the present invention is simple, sensitive, low-cost and convenient, with a minimum detection limit of 1 mg / kg for tetracycline antibiotics, and can perform in-situ detection on soils with a water potential range of -10.59 to -2.95 KPa.

[0029] The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil comprises the following steps: evenly spreading a soil sample to be tested adsorbed with tetracycline antibiotics on an LB solid culture medium containing E. coli pTGM fluorescent sensing bacteria, observing whether bright green fluorescent rings are generated on the soil surface and the culture medium surface after a short incubation, then detecting the fluorescence intensity of the fluorescent sensing bacteria and converting the bioavailability of adsorbed and desorbed tetracycline antibiotics in the soil through a standard curve of fluorescence value-concentration.

[0030] Wherein, the fluorescent sensing bacteria used can be E.coli pTGM fluorescent sensing bacteria, and the E.coli pTGM fluorescent sensing bacteria contain a pTGM plasmid carrying a tetracycline resistance gene tet (M), a transcriptional regulatory gene tetR, and a green fluorescent protein gene (gfp). When tetracycline penetrates into the cell interior of E.coli pTGM, the expression of the tetracycline resistance gene tet (M) is induced to provide ribosome protection, and at the same time, the tetR gene encoding the repressor protein will release the binding with PtetA, and then the reporter gene GFP connected downstream begins to express and produce green fluorescence. At the same time, combined with the growth inhibition of tetracycline on E.coli, the sensing bacteria can form a bright green fluorescent ring around the surface of the culture medium (containing desorbed tetracycline) and / or the surface of the soil (containing adsorbed tetracycline).

[0031] E.coli pTGM fluorescent sensing bacteria are spread on LB solid culture medium containing agar, and the soil sample to be tested is placed on the culture medium. After a short culture, observe whether a fluorescent circle is formed around the soil sample. Correspondingly, the bioavailability of antibiotics in the soil is positively correlated with the fluorescence intensity and the aperture diameter. The greater the fluorescence intensity and the larger the aperture diameter, the stronger the bioavailability of antibiotics in the soil sample. In order to quantitatively detect the bioavailability of antibiotics in the sample, the bacteria on the surface of the soil after culture and the bacteria on the surface of the culture medium can be extracted and their average fluorescence intensity can be detected respectively. The concentration of bioavailable antibiotics in the adsorbed and desorbed states in the soil can be calculated based on the fluorescence intensity.

[0032] For antibiotic substances adsorbed and / or desorbed by soil, water may affect the diffusion process of antibiotic molecules and thus affect their bioavailability; in this detection method, soil samples with a water potential range of -10.59 to -2.95 KPa can be tested.

[0033] The specific detection methods are as follows:

[0034] Step 1: Take the unknown soil to be tested, grind, sieve and sterilize it in sequence; preferably sieve it through a 2mm sieve;

[0035] Step 2: Add 2% (w / v) agar to LB medium, the specific components of which include 10.0 g / L NaCl, 10.0 g / L tryptone, and pH 7.0; add ampicillin after sterilization, and solidify after placing in a culture dish; spread E. coli TGM fluorescent sensing bacteria containing a pTGM plasmid carrying a tetracycline resistance gene tet (M), a transcriptional regulatory gene tetR, and a green fluorescent protein gene (gfp) on the culture dish;

[0036] Step 3: Place the soil sample on the culture medium, incubate it for a short time, and observe whether a fluorescent light ring is produced;

[0037] If you want to quantify the bioavailability of antibiotics in the sample, you need to proceed to step 4:

[0038] Step 4: Collect the bacterial flora on the soil sample and the aperture range culture medium sample respectively for extraction, and measure the average fluorescence intensity of each extracted E. coli cell by flow cytometry. According to the standard curve, the concentration of bioavailable antibiotics in the sample can be obtained. Laser confocal microscopy can be used to directly image the fluorescence intensity of the soil and agar surface. The E. coli pTGM colony is extracted by iotrolan density gradient separation method and the fluorescence intensity of each cell is measured by flow cytometry. In order to distinguish the content of antibiotics in the adsorbed state and the content of antibiotics in the desorbed state in the soil, the average fluorescence value of the bacteria attached to the soil part can be detected separately, which represents the content of antibiotics in the adsorbed state; or the average fluorescence value of the bacteria attached to the agar culture medium can be detected, which represents the content of antibiotics in the desorbed state.

[0039] In certain embodiments of the present invention, when a simple judgment or comparison is required as to whether a soil sample contains antibiotics, only the average fluorescence intensity or the diameter of the fluorescence circle needs to be compared; the average fluorescence intensity is positively correlated with the content of available antibiotics in the soil sample; the diameter of the fluorescence circle is positively correlated with the content of available antibiotics in the soil sample.

[0040] The biosensor system constructed by the above method is convenient, fast, highly sensitive, low-cost and environmentally friendly, and can realize the simultaneous detection of the bioavailability of tetracycline antibiotics in adsorbed and desorbed states in soil. It can also be applied to soils with a variety of different water potentials to simultaneously detect the bioavailability of antibiotics in adsorbed and desorbed states, providing important technical support for timely and effective monitoring of the behavior of antibiotics in the environment. The biosensor detection system provided by the present invention can also be applied to the detection of the bioavailability of other types of antibiotics, and can provide an effective solution for the detection and prevention of bioavailable antibiotics in the environment.

[0041] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0042] Example 1: Qualitative detection of bioavailability of tetracycline in soil

[0043] 1.1 Preparation of soil samples adsorbed with tetracycline

[0044] Three different types of soil were selected, namely clay loam, sandy clay loam and loamy sand. 15mL of tetracycline solution of different concentrations was transferred to glass centrifuge tubes containing 1g of different types of soil, so that the concentration of tetracycline reached 0.1-10mg / L (0, 0.1, 0.5, 1, 3, 7, 10mg / L). The above samples were placed on a shaker, taken out after shaking at room temperature for 24 hours, centrifuged at 3000rpm for 20 minutes, and most of the supernatant was removed. The remaining soil was freeze-dried, and the obtained soil was the soil sample adsorbed with tetracycline. At the same time, a soil sample for negative control, that is, a soil sample without tetracycline adsorption, was prepared.

[0045] 1.2 Preparation of biosensor system

[0046] Add 2% (w / v) agar to LB medium to obtain LB solid medium. Sterilize the above medium and cool it to 50-60°C, then add ampicillin to make the final concentration of ampicillin in the medium 100 mg / L. Then, take 7 mL of the medium while it is hot and transfer it to a sterile culture dish, and set it aside after solidification.

[0047] E. coli pTGM fluorescent sensing bacteria contain the pTGM plasmid carrying the tetracycline resistance gene tet (M), the transcriptional regulatory gene tetR and the green fluorescent protein gene (gfp). E. coli pTGM fluorescent sensing bacteria were provided free of charge by Professor Sorensen of the University of Copenhagen, Denmark (references: Bahl MI, Hansen LH, SJ. Construction of an extended range whole-cell tetracycline biosensor by use of the tet(M)resistance gene[J]. FEMS Microbiology Letters, 2005, 253(2): 201-205.), the tetracycline resistance gene tet(M) was inserted into a pTGM plasmid containing a tetracycline-inducible promoter, a transcriptional regulatory gene tetR and a green fluorescent protein gene (gfp), and the plasmid was then transformed into E. coli to obtain E. coli pTGM fluorescent sensing bacteria.

[0048] E. coli pTGM was cultured in LB liquid medium (containing 10.0 g / L NaCl, 10.0 g / L tryptone, pH 7.0) containing 100 mg / L ampicillin. After culturing at 30°C and 150 rpm for 8 hours, 100 μL of the bacterial culture of E. coli pTGM was evenly spread on LB solid medium containing 100 mg / L ampicillin when the bacteria grew to the exponential growth phase.

[0049] 1.3 Observe whether the soil adsorbed with tetracycline produces a bright fluorescence ring in the biosensor system

[0050] After freeze-drying the soil with a certain amount of tetracycline adsorbed on the surface, 10 mg was placed on a solid culture medium inoculated with E. coli pTGM. After inverted culture at 30°C for 8 hours, the growth of E. coli pTGM on the plate was observed under UV light.

[0051] The results are as follows Figure 1 As shown, 0 corresponds to 0 mg / L of tetracycline added during soil treatment, 1 corresponds to 0.1 mg / L of tetracycline added during soil treatment, 2 corresponds to 0.5 mg / L of tetracycline added during soil treatment, 3 corresponds to 1 mg / L of tetracycline added during soil treatment, 4 corresponds to 3 mg / L of tetracycline added during soil treatment, 5 corresponds to 7 mg / L of tetracycline added during soil treatment, and 6 corresponds to 10 mg / L of tetracycline added during soil treatment; when the initial concentration of tetracycline is 7 mg / L (label 5) and 10 mg / L (label 6), bright fluorescent rings can be clearly observed around the three soils, indicating that bioavailable tetracycline exists in the soil. Compared with the negative control, no fluorescent ring is observed around it.

[0052] Example 2: Detection of the bioavailability of tetracycline in soil

[0053] 2.1 Relationship between fluorescence intensity and bioavailable tetracycline concentration in soil

[0054] 15 ml of tetracycline solution at 0, 0.1, 0.5, 1, 3, 7, and 10 mg / L was used to treat 1 g of soil, and the soil was inoculated on LB solid medium containing 100 mg / L ampicillin coated with 100 μL of bacterial culture of E.coli pTGM according to the method of Example 1, and incubated at 30°C for 8 h.

[0055] The Histodenz density gradient dilution method was used to extract bacteria growing on the surfaces of three types of soil, namely clay loam, sandy clay loam and loamy sand, in the culture plates after culture. PBS buffer was used to extract bacteria growing on the agar surface within 0.75 cm from the center of the soil (fluorescence circle range). The average fluorescence intensity of each E. coli pTGM cell was measured using a BDLSRII flow cytometer for the bacteria extracted from the soil surface and agar surface.

[0056] Construct a soil adsorption experiment. First, 15 mL of tetracycline solution of different concentrations was filtered and sterilized, and then transferred to a 30 mL sterile glass centrifuge tube containing 1 g of sterilized soil, and the concentration of tetracycline was adjusted to between 0.1-10 mg / L. The prepared solution needs to be kept away from light to reduce its photolysis reaction due to light. According to the results of the preliminary experiment, it is known that within 24 hours, the soil and tetracycline in the solution can reach adsorption equilibrium. The adsorption time was set to 24 hours, and two repeated experiments were set for each treatment to ensure the accuracy of the results. All samples were placed on a shaker and taken out after shaking at room temperature for 24 hours. Subsequently, centrifuged at 3000 rpm for 20 minutes, and 1 mL of the supernatant was taken for liquid chromatography-tandem mass spectrometry (LC / MS / MS) determination. By calculating the difference between the initial concentration of tetracycline in the solution and the final concentration after 24 hours, we can derive the amount of tetracycline adsorbed by the soil.

[0057] The fluorescence detection results in the culture medium and soil of the experimental samples corresponding to each tetracycline treatment concentration were matched one by one with the corresponding soil adsorbed tetracycline concentration to prepare Figure 2 The results are as follows Figure 2As shown, taking clay loam as an example, when the fluorescence intensity of bacteria isolated from the soil surface was 827±233, 3958±419, 13845±3204, 63922±16224, 115587±13521 and 134932±5346, the corresponding bioavailable tetracycline concentrations in the soil were 1.4±0.0, 7.4±0.0, 14.9±0.0, 44.7±0.0, 104.4±0.0 and 148.9±0.2 mg / kg. It can be seen that the fluorescence intensity detected is positively correlated with the concentration of bioavailable tetracycline in the soil.

[0058] In addition, the presence of tetracycline in soil samples includes adsorption and desorption. For the soil sample culture medium after cultivation, the fluorescence detection results on the soil represent the content of tetracycline adsorbed in the soil, and the fluorescence detection results on the culture medium represent the content of tetracycline desorbed in the soil. Through this method, the concentration values ​​of adsorbed antibiotics in the soil samples to be tested can be detected separately, and the concentration values ​​of desorbed antibiotics can also be detected.

[0059] 2.2 Relationship between fluorescence ring diameter and bioavailable tetracycline concentration in soil

[0060] 15 ml of tetracycline solution at 5, 10, 30 and 50 mg / L was used to treat 1 g of soil. According to the method of Example 1, the soil was inoculated on LB solid medium containing 100 mg / L ampicillin coated with 100 μL of bacterial culture of E.coli pTGM. After cultivation, the results were as follows: Figure 3 shown.

[0061] like Figure 3 As shown in the figure, in the same period of time, when the adsorption of tetracycline by the three soils increased (the process was the same as the soil adsorption experimental steps mentioned above), a clearer and larger diameter fluorescent ring was observed under UV light, and the diameter of the fluorescent ring increased with the increase of the amount of tetracycline adsorbed by the soil. Taking loam sand as an example ( Figure 3 When the adsorption amount of tetracycline was 85.47, 160.43, 459.80 and 757.11 mg / kg, the diameters of the fluorescence rings produced were about 0.98, 1.36, 1.66 and 2.01 cm, respectively. It can be seen that the concentration of bioavailable tetracycline in the soil is positively correlated with the diameter and intensity of the fluorescence ring.

[0062] Example 3: Effect of water potential on the detection of antibiotic substances adsorbed and / or desorbed from soil.

[0063] For antibiotic substances adsorbed and / or desorbed by soil, water may affect the diffusion process of antibiotic molecules and thus affect their bioavailability. This example further studies the effect of soil water potential on antibiotic bioavailability.

[0064] Prepare medium containing different water content (add different amounts of PEG to change the water condition of the medium) to simulate soil with different water potential. 20 ml of polyethylene glycol (PEG) solution of different concentrations were measured and poured onto the same volume of solidified LB medium and left for 24 hours so that PEG can diffuse into the solid medium to the greatest extent, thereby reducing the water potential in the medium. After 24 hours, remove the PEG remaining on the surface of the medium, then perform an agar diffusion experiment, and measure the fluorescence intensity of E. coli grown on the soil surface after 12 hours of cultivation, as shown in Example 1.

[0065] The fluorescence intensity of soils with different water potentials and the corresponding E. coli pTGM fluorescent sensing bacteria is shown in Figure 2. Figure 4 As shown, the results show that with the decrease of soil water potential, the fluorescence intensity of E. coli pTGM fluorescent sensing bacteria shows a downward trend, but the biosensor detection system constructed by the present invention can still achieve better detection effect on soil with a water potential range of -10.59 to -2.95 Kpa.

[0066] The above phenomena all demonstrate that the E. coli pTGM biosensor detection system constructed by the present invention can quickly and sensitively detect the bioavailability of tetracycline in soil. The intensity and diameter range of the fluorescence ring directly reflect the bioavailable antibiotics in the soil, and can provide timely and effective early warning for preventing the generation and spread of antibiotic resistance genes in soil microbial communities.

[0067] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A biosensor detection system, characterized in that: The invention comprises a solid culture medium containing agar and containing a screening antibiotic, and E. coli pTGM fluorescent sensor bacteria coated on the solid culture medium containing agar, wherein the E. coli pTGM fluorescent sensor bacteria can respond to the screening antibiotic.

2. The biosensor detection system according to claim 1, characterized in that: E. colipTGM fluorescent sensing bacteria contain the pTGM plasmid carrying the tetracycline resistance gene tet (M), the transcriptional regulatory gene tetR and the green fluorescent protein gene (gfp).

3. The biosensor detection system according to claim 2, characterized in that: The culture medium is LB solid culture medium containing 100 mg / L ampicillin and 2% (w / v) agar.

4. The biosensor detection system according to claim 3, characterized in that: Cultivate E. coli TGM fluorescent sensing bacteria to OD 600 When the concentration is 0.4-0.6, apply it on the culture medium.

5. A method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil using the biosensor detection system according to any one of claims 1 to 4, characterized in that: The soil sample to be tested is placed on a solid culture medium. After incubation, whether fluorescent substances are formed around the soil sample is observed to determine whether the soil sample contains bioavailable antibiotics.

6. The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil according to claim 5, characterized in that: The antibiotic is a tetracycline antibiotic, preferably one or more of tetracycline, oxytetracycline and chlortetracycline; Detection of adsorbed bioavailable antibiotics and / or desorbed bioavailable antibiotics in soil.

7. The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil according to claim 5, characterized in that: The specific method is as follows: Step 1: Take the unknown soil to be tested, grind, sieve and sterilize it in sequence; Step 2: Add 2% (w / v) agar to LB medium, add ampicillin to 100 mg / L after sterilization, and place in a culture dish to solidify; spread E. coli pTGM fluorescent sensor bacteria containing pTGM plasmid carrying tetracycline resistance gene tet (M), transcriptional regulatory gene tetR and green fluorescent protein gene (gfp) on the culture dish; Step 3: Place the soil sample on the culture medium and observe whether fluorescent apertures are produced after incubation to determine whether the soil sample contains bioavailable antibiotics.

8. The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil according to claim 7, characterized in that: Also includes step four; Step 4: Collect the bacterial flora on the soil sample and the aperture range culture medium sample respectively for extraction, detect and calculate the average fluorescence intensity, and obtain the concentration of bioavailable antibiotics in the sample based on the standard curve.

9. The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil according to claim 8, characterized in that: The soil and agar surfaces can be imaged directly using laser confocal microscopy to detect the fluorescence intensity of the formed fluorescent substances; Alternatively, E. coli pTGM colonies were extracted using iotrolan density gradient separation and the fluorescence intensity of each cell was measured using flow cytometry.

10. The method for simultaneously detecting the bioavailability of adsorbed and desorbed antibiotics in soil according to claim 5, characterized in that: The water potential range of the soil samples to be tested is -10.59 to -2.95 Kpa.

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