CN114509420B - Method for evaluating migration risk of antibiotic resistance gene - Google Patents

Abstract

The invention discloses a method for evaluating the risk of antibiotic resistance gene migration. The method comprises: providing a first target bacterial strain and a second target bacterial strain, wherein the first target bacterial strain has dual fluorescent signals, and the second target bacterial strain has multi-antibiotic sensitivity; The zygote is contacted under conditions suitable for conjugation; the zygote is subjected to fluorescence detection. The method of the present invention can quickly assess the risk of antibiotic resistance gene migration, effectively solve the time-consuming and labor-intensive defects in existing methods for calculating migration frequency, and rapidly assess the risk of horizontal migration of drug resistance genes under different environmental conditions. In addition, the double fluorescent drug-resistant Escherichia coli constructed by the present invention is used as a donor bacterium for studying the horizontal conjugative transfer of antibiotic resistance genes, and can also quickly locate the drug-resistant genes entering the intestinal tract of plants or aquatic organisms, thereby realizing the drug-resistant genes. Migration visualization.

Description

A method to assess the risk of transfer of antibiotic resistance genes

technical field

The invention relates to the field of biotechnology, in particular to a method for assessing the migration risk of antibiotic resistance genes.

Background technique

With the widespread use of antibiotics, the number of antibiotic-resistant bacteria driven by antibiotic resistance genes (ARGs) is increasing, and other non-resistant bacteria can also acquire resistance through conjugation, transduction, and transformation. The water environment is usually a natural reservoir of drug-resistant bacteria and drug-resistant genes, and an important environmental medium for the preservation, amplification, transfer and diffusion of drug-resistant genes. A large number of reports have shown that hundreds of drug resistance genes or even multiple drug resistance genes have been detected in various surface waters, including hospital sewage, domestic sewage, sewage treatment plants, groundwater and even drinking water.

The traditional horizontal migration research of drug-resistant genes uses filter membranes or liquid incubation methods to screen zygotes in liquid-solid medium containing antibiotic selection pressure for dilution and counting. Although the whole process is simple to operate, it is time-consuming. It is long and cannot be visualized, so it is impossible to quickly analyze the migration risk of drug resistance genes under different water quality environmental conditions.

Contents of the invention

In order to solve the technical problems in the prior art, the present invention constructs two-color fluorescent Escherichia coli carrying drug resistance, and mixes the two-color fluorescent Escherichia coli with bacteria sensitive to various antibiotics and water samples to be tested, The bacterial fluorescence signal was measured by multi-dimensional panoramic flow cytometer, and the horizontal migration frequency of ARG was calculated according to the results of the number of bacteria measured by different fluorescent signals. Specifically, the present invention includes the following contents.

The first aspect of the present invention provides a method for assessing the risk of antibiotic resistance gene migration, which includes:

(1) providing a first target bacterial strain and a second target bacterial strain, wherein the first target bacterial strain has dual fluorescent signals, and the second target bacterial strain has multiple antibiotic sensitivity;

(2) contacting the first target strain with the second target strain in the sample to be tested under conditions suitable for conjugation to obtain zygotes;

(3) Perform fluorescence detection on the zygote.

According to the method for assessing the risk of antibiotic resistance gene transfer in the present invention, preferably, the first target strain is constructed by conjugating a recipient bacterium marked with red fluorescent protein and a donor bacterium marked with green fluorescent protein.

According to the method for assessing the risk of antibiotic resistance gene transfer in the present invention, preferably, the donor bacteria and the recipient bacteria are Escherichia coli.

According to the method for assessing the risk of antibiotic resistance gene transfer in the present invention, preferably, the Escherichia coli includes Escherichia coli K12 or Escherichia coli DH 5α.

According to the method for assessing the migration risk of antibiotic resistance genes of the present invention, preferably, the donor bacteria have antibiotic resistance or carry drug resistance genes.

According to the method for assessing the risk of transfer of antibiotic-resistant genes according to the present invention, preferably, the antibiotics include at least one or a combination of tetracycline, ampicillin and kanamycin; the drug-resistant genes include: tetA, At least one or a combination of tnpR and aphA.

According to the method for assessing the migration risk of antibiotic-resistant genes according to the present invention, preferably, the frequency of horizontal migration of antibiotic-resistant genes=(green fluorescent signal channel bacterial detection amount-green fluorescent signal channel:red fluorescent signal channel bacterial detection amount )/(total amount of bacteria-detection amount of bacteria in the red fluorescent signal channel).

According to the method for assessing the risk of antibiotic resistance gene migration in the present invention, preferably, the conditions suitable for conjugation refer to temperature of 34-39° C., 150-200 rpm, and time of 5-12 h.

According to the method for assessing the migration risk of antibiotic resistance genes described in the present invention, preferably, a flow cytometer is used for fluorescent signal detection.

A second aspect of the present invention provides a system for assessing the risk of antibiotic resistance gene migration, which includes:

A first target strain with dual fluorescent signals;

A second target strain, wherein the second target strain is used for gene transfer of Escherichia coli, and the second target strain has multiple antibiotic sensitivity; and

Multi-channel fluorescence detection device.

The present invention also provides the use of reagents in the preparation of kits for assessing the risk of antibiotic resistance gene migration by the following method, wherein the method includes: (1) providing a first target strain and a second target strain, wherein the first The target strain has dual fluorescent signals, and the second target strain has multi-antibiotic sensitivity; (2) contacting the first target strain with the second target strain in the sample to be tested under conditions suitable for conjugation, Obtain the zygote; (3) carry out fluorescence detection to the zygote;

The reagents include: a first target strain with dual fluorescent signals;

A second target strain, wherein the second target strain is used for gene transfer of Escherichia coli, and the second target strain has multiple antibiotic sensitivity; and

A culture solution for cultivating the first strain or the second strain, the culture solution includes 0.01-0.5 mM phosphate buffer, preferably 0.1 mM phosphate buffer.

The technical effects of the present invention include but are not limited to:

(1) The method of the present invention can quickly assess the risk of antibiotic resistance gene migration under various water quality conditions (pH, conductivity, COD, antibiotics and other organic substances) to be tested, effectively solving the existing method for calculating migration frequency The defects of time-consuming and labor-intensive methods exist, and at the same time, it can quickly assess the risk of horizontal transfer of drug-resistant genes under different environmental conditions.

(2) The present invention uses bacteria carrying double fluorescence and sensitive bacteria to detect the risk of antibiotic resistance gene migration under the water sample to be tested, by constructing bacteria carrying double fluorescence as donor bacteria, sensitive bacteria as acceptor bacteria, acceptor and donor After the zygote is conjugated in the water sample to be tested, the zygote can be quickly quantified by collecting a large number of bacteria and detecting the fluorescent signal, which can quickly assess the horizontal migration risk of ARG in various water quality conditions to be tested, and can be applied to the field of sewage risk assessment .

(3) The fluorescent expression of the dual fluorescent drug-resistant Escherichia coli constructed by the present invention is stable, the expression of green fluorescent light will not be lost due to the migration of the plasmid, and no inducer is needed to induce fluorescent expression; the screened Aeromonas hydrophila It has high sensitivity to drug resistance genes; the multi-dimensional panoramic flow cytometer has high response signals to red and green fluorescence, and can quickly assess the risk of antibiotic resistance gene migration under different water quality conditions of the water samples to be tested.

(4) The double fluorescent drug-resistant Escherichia coli constructed by the present invention is used as the donor bacteria for studying the horizontal conjugation transfer of antibiotic resistance genes. , Rapid calculation of horizontal splice frequency of drug resistance genes in the gut of aquatic organisms.

(5) The double-fluorescent drug-resistant Escherichia coli constructed by the present invention is used as the donor bacteria for studying the horizontal splice transfer of antibiotic-resistant genes, and can even quickly locate the drug-resistant genes entering the intestinal tract of plants or aquatic organisms through fluorescent signals, Realize the visualization of drug resistance gene migration.

Description of drawings

Figure 1 is a photograph of the colony growth morphology of the bicolor Escherichia coli constructed in the embodiment of the present invention.

Fig. 2 is a scanning image of the double-color Escherichia coli constructed in the embodiment of the present invention under a laser confocal scanning microscope.

Fig. 3 is a flow cytometry diagram provided by an embodiment of the present invention after co-cultivating two-color Escherichia coli and sensitive Aeromonas hydrophila in the water sample to be tested.

Fig. 4 is a diagram of calculation results of the conjugation frequency of antibiotic resistance genes in different water samples to be tested provided by the embodiment of the present invention.

detailed description

Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and every intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control. "%" is a percentage by weight unless otherwise specified.

The method for assessing the migration risk of antibiotic resistance genes of the present invention includes steps (1)-(3), which will be described in detail below.

step 1)

The first target strain of the present invention has dual fluorescence signals, which means having two different detectable fluorescences, preferably red fluorescence and green fluorescence. The dual fluorescent signal strain was obtained by mating the recipient bacteria with red fluorescent protein and the donor bacteria with green fluorescent protein. The conjugation method is not particularly limited, and conjugation methods known in the art can be used, examples of which include, but are not limited to, solid plates, liquid static culture, liquid shaker culture (filtrate conjugation method) and the like.

Preferably, Escherichia coli K12 carrying red fluorescent protein in the genome is selected as the recipient bacterium, and Escherichia coli DH 5α carrying green fluorescent protein is selected as the donor bacterium. The gene expressing the fluorescent protein can be carried by the genome of the host bacterium or be operably linked to the gene through an expression vector. In some specific embodiments, the genome of the recipient bacteria carries a gene expressing red fluorescent protein. In another embodiment, the donor bacteria contain a plasmid carrying a gene for expressing green fluorescent protein. The type of plasmid is not particularly limited. Preferably, the plasmid used in the present invention is RP4 plasmid, which has a wide host range and high mobility.

Preferably, the fluorescent bacterium K12 is labeled with mCherry red fluorescent protein, and the fluorescent bacterium DH 5α is labeled with sfgfp green fluorescent protein.

The terms "operably linked" and "label" used in the present invention have meanings commonly understood in the art.

In the present invention, the donor bacteria in the first target strain has drug resistance, drug resistance means that it carries at least two of the three antibiotic resistances selected from tetracycline, ampicillin and kanamycin, for example, it can be simultaneously Carry the above three antibiotic resistance. Preferably, the donor bacterium in the first target strain carries at least two drug resistance genes selected from tetA, tnpR, and aphA, for example, it may carry the above three drug resistance genes at the same time.

In the present invention, the donor bacterium: the recipient bacterium: LB medium = 1:1-2:1-5 is used for conjugation, and the conjugation time is 5-12 h. Preferably, the donor bacteria: the recipient bacteria: LB medium = 1:1-1.5:2-4, and the conjugation time is 8-12 hours. Also preferably, the donor bacteria: the recipient bacteria: LB medium = 1:1:3, and the conjugation time is 12 hours. In the present invention, the bonding time does not exceed 12 hours, which greatly shortens the bonding experiment time.

Preferably, the culture temperature of the donor bacteria, recipient bacteria and conjugation fluid is 34-41°C, more preferably 35-39°C, further preferably 36-38°C. The culture condition for conjugation is 140 rpm-220 rpm constant temperature shaker, preferably 150 rpm-210 rpm, and also preferably 160 rpm-200 rpm.

In the present invention, after pure culture, wash, resuspend and dilute until the numbers of the donor bacteria and the recipient bacteria respectively reach 108-10 CFU/mL, preferably 109-10 CFU/mL, and more preferably 1010 CFU/mL.

It should be noted that in the process of conjugating the donor bacteria and the recipient bacteria, red and green fluorescence do not need to add inducers to induce luminescence, and the fluorescence expression of the constructed double fluorescent drug-resistant Escherichia coli is stable. The expression of green fluorescence will not be lost due to the migration of the plasmid.

Preferably, the green fluorescent protein (sfgfp) is measured with an excitation light wavelength of 488 nm, and the red fluorescent protein (mCherry) is measured with an excitation light wavelength of 581 nm. The instrument for measuring fluorescence intensity is not particularly limited, and devices or devices known in the art can be used, examples of which include but are not limited to, for example, a laser confocal scanning microscope.

The second target strain of the present invention has multiple antibiotic susceptibility. Antibiotic susceptibility can be identified by drug-sensitive tablets, examples of which include, but are not limited to: oxytetracycline, roxithromycin, sulfonamides, kanamycin, ampicillin, and amoxicillin.

In the present invention, the source of the second target strain is not particularly limited, and it can be any antibiotic-sensitive bacteria from any sample. For example, it can be indigenous flora from water bodies, soil. The types of water bodies to be tested include but are not limited to surface water, aquaculture wastewater or water bodies containing different pollutants (such as urban sewage, medical wastewater, various self-prepared aqueous solutions, etc.). In a specific embodiment, the second target strain of the present invention is Aeromonas hydrophila.

Preferably, the second target strain of the present invention is a bacterium that is isolated from the feces and water mixture of Xenopus tropicalis and is sensitive to various antibiotics and can perform gene migration with Escherichia coli.

step (2)

In step (2), the first target strain is contacted with the second target strain in the sample to be tested under conditions suitable for conjugation to obtain zygotes.

Preferably, the sample to be tested is sterilized by filtration. The filter sterilization step is not particularly limited, for example, the sample to be tested can be sterilized through a filter membrane with a specific pore size, and the pore size of the filter membrane is usually 0.22 μm or smaller.

Conditions suitable for conjugation mean that the temperature of the mixed culture is 34-41°C, preferably 35-39°C, more preferably 36-38°C. The culture condition for conjugation is 140 rpm-220 rpm constant temperature shaker, preferably 150 rpm-210 rpm, and also preferably 160 rpm-200 rpm. Joining time is 8-15 h. Also preferably 8-12h.

Preferably, the bacterial counts of the first target strain and the second target strain are preset to be 109, 1010, and 1011 CFU/mL.

The sample to be tested in the present invention refers to a sample from any source, including but not limited to a water sample from a water body or a soil sample from soil. It can be understood that the sample to be tested can be derived from any plant or animal sample of the above-mentioned water body or soil.

step (3)

In step (3), a specific wavelength laser is used for flow cytometry fluorescence quantification. The type of flow cytometer is not particularly limited as long as it has multiple fluorescence channels.

In the present invention, the migration frequency of the drug-resistant gene in the sample to be tested can be calculated according to the results of the amount of bacteria measured by different fluorescent channels of the flow cytometer. Flow cytometry is used to identify fluorescence, which can be accurately identified and counted and sorted according to the different fluorescent colors of donors, acceptors, and zygotes.

Preferably, when measuring the fluorescence result in step (3), perform gradient dilution to select the optimal concentration to collect the fluorescence result, such as pretreatment of the sample on the machine according to the absorbance of OD600 nm, and the preset range is 0.5-0.7. More preferably, in step (3), by measuring the fluorescence signal of Escherichia coli K12 and the fluorescence signal of Escherichia coli DH5α to exclude single fluorescence interference, the amount of bacteria collected is preset as 5000-10000, and the bacteria collected are displayed in the form of fluorescence scatter distributed on the axis.

In the step (3) of the present invention, according to the fluorescent signal ratio that different channels collect, calculate conjugation frequency, preferably, the frequency of the horizontal migration of antibiotic resistance gene=(green fluorescent signal channel bacterial detection amount-green fluorescent signal channel:red fluorescence signal channel bacteria detection amount)/(total amount of bacteria-red fluorescent signal channel bacteria detection amount). In a specific embodiment, the Ch02 channel collects the amount of bacteria with sfgfp fluorescent protein positive signal, the Ch06 channel collects the bacterial amount of mCherry fluorescent protein positive channel, and the dual-fluorescent bacteria show fluorescent signals at the same time in the Ch02 and Ch06 channels, and the zygote is in the Ch02 channel A fluorescent signal appears, and the frequency of horizontal migration of ARG=(detected amount of bacteria Ch02-Ch02: detected amount of bacteria Ch06)/(total amount of bacteria-detected amount of bacteria Ch06).

In the present invention, the conjugation fluid obtained in step (2) can be directly counted by flow cytometry without multiple cultures, which ensures the initial growth status of the zygote in the original conjugation environment, and can effectively collect some of the zygotes that cannot be cultured. The number of zygotes increases the accuracy of the frequency of zygoses. In addition, the zygote includes not only the culturable part of the flora, but also the non-culturable part of the flora, the calculated conjugation frequency is more accurate, and the experimental error caused by multiple cultures is reduced.

Those skilled in the art should understand that other steps or operations may also be included before and after the above steps (1)-(3), or between any of these steps, such as further optimizing and/or improving the method of the present invention.

The double-fluorescent drug-resistant Escherichia coli constructed by the present invention is used as the donor bacteria for studying the horizontal conjugation transfer of antibiotic resistance genes, and can even quickly locate the drug-resistant genes entering the intestinal tract of plants or aquatic organisms through fluorescent signals, so as to realize resistance Pharmacogene migration visualization.

Example

This example is a method for assessing the risk of antibiotic resistance gene transfer, as follows.

(1) Take out red fluorescent Escherichia coli K12 and green fluorescent Escherichia coli DH5α from the -80°C refrigerator, apply after activation, and check whether the fluorescent proteins of red fluorescent Escherichia coli and green fluorescent Escherichia coli are normally expressed.

(2) Select the above two strains of Escherichia coli and culture them to logarithmic growth phase in LB culture medium, in which the LB culture medium for cultivating Escherichia coli DH 5α contains 100 μg/mL ampicillin, 50 μg/mL kanamycin and tetracycline hydrochloride at 10 μg/mL.

(3) Wash and dilute Escherichia coli K12 and Escherichia coli DH 5α with physiological saline or sterile phosphate buffer (0.1 mM) until the number of bacteria reaches 1010 CFU/mL respectively to obtain a preconjugated bacterial solution.

(4) Red Escherichia coli K12 was used as the recipient bacterium, green Escherichia coli DH 5α was used as the donor bacterium, and the conjugation was carried out according to the ratio of donor bacterium:recipient bacterium:LB medium=1:1:3, and the overall conjugation system was designed as 20 mL, the binding time is 12 h.

(5) Take conjugation fluid and apply it to LB solid medium. After 18 hours of culture, use an inverted fluorescence microscope to check the fluorescence signal of each single colony, and select colonies with double fluorescence signals.

(6) The selected double fluorescent signal colonies were subjected to secondary LB liquid expansion, and 2 mL of the bacterial liquid was pipetted into a 24-well cell culture plate, and the fluorescent signal was observed with an inverted fluorescence microscope.

(7) Select the bacterial solution in which the red and green fluorescent signals are stable, and streak the plate to separate single colonies, as shown in Figure 1.

(8) Observe the fluorescence signal of the isolated single colony with an inverted fluorescence microscope, and repeat it several times to screen out the double-color fluorescent E. coli with the best fluorescence signal and the best growth conditions as the target strain, as shown in Figure 2, Figure 2 The medium two-color fluorescent E. coli showed stable fluorescent signals at both 488 nm and 581 nm excitation wavelengths.

(9) Draw 10 mL of feces-water mixture from the tropical Xenopus frog culture tank, gently absorb the supernatant after high-speed centrifugation, and use a pipette to draw 1 mL of the precipitate into the LB medium, 37 ° C, 180 rpm Incubate under the condition for 24 h.

(10) After the above bacterial solution was serially diluted, 100 μL was applied to LB solid medium and cultured at 37°C for 24 hours.

(11) Select a single colony with different colony morphology characteristics in the petri dish for LB liquid expansion, and use the drug-sensitive tablets of oxytetracycline, roxithromycin, sulfonamide, kanamycin, ampicillin, and amoxicillin to detect The sensitivity of different bacterial solutions, and finally select the bacteria that are sensitive to multiple antibiotics for bacterial species identification, and determine that it is Aeromonas hydrophila.

(12) Wash and adjust the overnight cultured two-color fluorescent bacteria (donor bacteria) and Aeromonas hydrophila (recipient bacteria) with normal saline, centrifuge at 8000xg for 6 min to collect the precipitate, and resuspend the collected bacteria in LB medium for use. The number of bacteria reached 1010CFU/mL.

(13) Take the Pearl River water, artificial lake water, lake water and disinfection effluent as water samples to be tested, and deionized water as a blank control. The water samples to be tested are treated with a 0.22 μm organic filter membrane for sterilization, and the membrane water to be tested is obtained. Sample.

(14) Take 4 mL each of the adjusted two-color fluorescent bacteria (donor bacteria) and Aeromonas hydrophila (recipient bacteria) and add 17 mL of each membrane-passed water sample to be tested (including 5 mL LB culture solution) cultured in a constant temperature shaker at 37°C±2°C and 180 rpm for 12 hours to obtain conjunctive fluid.

(15) Take 8 mL of the above junction solution and centrifuge at 8000xg for 8 min in a centrifuge, discard the filtrate, wash the bacterial solution with 8 mL phosphate buffer (0.1 mM) repeatedly for 3 times, and adjust to OD600nm≈0.7.

(16) After the above-mentioned pretreated conjugation fluid was sieved with 70 μm sterile cells, collect 1 mL of the bacterial liquid in a 1.5 mL centrifuge tube, collect fluorescence signals by flow cytometry, and set the total number of collected cells to 10,000. The bacterial count of green fluorescent protein (sfgfp) signal was collected in Ch02 channel with 488 nm excitation light wavelength, and the bacterial count of red fluorescent protein (mCherry) signal was collected in Ch06 channel with 581 nm excitation light wavelength.

Figure 3 shows the flow cytometric imaging results after adding two-color Escherichia coli and sensitive Aeromonas hydrophila to the water sample to be tested. In Figure 3, Aeromonas hydrophila was used as the recipient bacteria in Ch02 and Ch06 There was no fluorescent signal in any channel, and the two-color fluorescent bacteria as the donor bacteria showed obvious green and red fluorescent signals in the Ch02 and Ch06 channels respectively; after bacterial co-cultivation, the two-color fluorescent bacteria lost the green fluorescent signal in the Ch02 channel due to the migration of the plasmid, Only a single red fluorescent signal appears in the Ch06 channel, while Aeromonas hydrophila shows a separate green fluorescent signal in the Ch02 channel due to the obtained plasmid, which is the zygote.

(17) Frequency of horizontal migration of ARG=(Ch02 bacterial detection amount-Ch02:Ch06 bacterial detection amount)/(Total amount of bacteria-Ch06 bacterial detection amount), draw migration risk calculation results in Figure 4. Figure 4 shows that compared with other surface water and disinfection effluent, deionized water has the highest migration frequency of resistance genes; compared with surface water, sterilized water also shows a higher conjugation frequency, surface water, artificial lake water resistance gene The migration frequency was the lowest, as expected. And according to the different comprehensive water quality conditions, the migration frequency detection difference is significant, indicating that the method of the present application can quickly evaluate the antibiotic resistance genes under the water quality conditions (pH, conductivity, COD, antibiotics and other organic substances) of various water samples to be tested risk of migration.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various adaptations or changes may be made to the illustrated exemplary embodiments of the invention without departing from the scope or spirit of the invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims (2)

1. A method for evaluating the migration risk of antibiotic drug resistance genes of water samples to be detected with various water qualities under different environmental conditions is characterized by comprising the following steps of:

(1) Providing a first target strain and a second target strain, wherein the first target strain has a bifluorescent signal, the second target strain is aeromonas hydrophila with multiple antibiotic susceptibility, the first target strain is a recipient strain of escherichia coli K12 whose genome carries mCherry red fluorescent protein, an donor strain of escherichia coli DH5 alpha carrying sfgfp green fluorescent protein is selected, and the donor strain: the recipient bacterium: LB broth =1:1-2:1-5, the jointing time is 5-12h, the culture temperature of the donor bacteria, the acceptor bacteria and the jointing liquid is 34-41 ℃, and the culture condition for jointing is a constant temperature shaking table at 140-220 rpm; the donor bacterium has antibiotic resistance or carries a drug-resistant gene;

(2) The first target strain and the second target strain are resuspended by LB culture solution to make the number of bacteria reach 10 ¹⁰ CFU/mL, enabling the first target strain to contact with the second target strain in a water sample to be detected after being subjected to membrane filtration sterilization by an organic filter membrane under the condition suitable for conjugation to obtain a conjugant, wherein the condition suitable for conjugation is that the conjugation temperature is 34-39 ℃, the constant temperature shaking table at 150-200 rpm is adopted, and the conjugation time is 5-12h;

(3) Performing fluorescence detection on the zygote processed in the step (2) by adopting a flow cytometer;

the frequency of horizontal migration of antibiotic resistance genes = (green fluorescent signal channel bacterial detection amount-green fluorescent signal channel bacterial detection amount: red fluorescent signal channel bacterial detection amount)/(total amount of bacteria-red fluorescent signal channel bacterial detection amount).

2. The method for evaluating the migration risk of the antibiotic resistance genes of the water samples to be tested with various water qualities under different environmental conditions according to claim 1, wherein the antibiotic comprises at least one of tetracycline, ampicillin and kanamycin, or a combination thereof; the drug resistance gene includes at least one of tetA, tnPR, and aphA or a combination thereof.

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