CN114509420B - Method for evaluating migration risk of antibiotic resistance gene - Google Patents
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Abstract
The invention discloses a method for evaluating migration risk of antibiotic resistance genes. The method comprises the following steps: providing a first target strain and a second target strain, wherein the first target strain has a bifluorescent signal and the second target strain has a multiple antibiotic susceptibility; contacting the first target strain with the second target strain in a sample to be tested under conditions suitable for conjugation to obtain a zygote; the zygotes were subjected to fluorescence detection. The method can quickly evaluate the migration risk of the antibiotic drug-resistant gene, effectively overcomes the defects of time and labor waste in the existing migration frequency calculation method, and simultaneously quickly evaluates the horizontal migration risk of the drug-resistant gene under different environmental conditions. In addition, the bifluorescence drug-resistant escherichia coli constructed by the invention is used as a donor bacterium for researching horizontal joint transfer of antibiotic drug-resistant genes, and can also be used for rapidly positioning the drug-resistant genes entering the intestinal tracts of plants or aquatic organisms, so that the drug-resistant gene migration visualization is realized.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for evaluating migration risk of antibiotic resistance genes.
Background
With the widespread use of antibiotics, the number of antibiotic-resistant bacteria driven by Antibiotic Resistance Genes (ARG) is increasing, and other non-resistant bacteria can also be made resistant by conjugation, transduction, transformation, and the like. The water environment generally becomes a natural storage base of drug-resistant bacteria and drug-resistant genes and is an important environmental medium for the preservation, amplification, transfer and diffusion of the drug-resistant genes. A large number of reports show that hundreds of drug-resistant genes and even multiple drug-resistant genes are detected in various types of surface water, including hospital sewage, domestic sewage, sewage treatment plants, underground water and even drinking water.
The traditional horizontal migration research of the drug-resistant genes is to screen the zygotes in a liquid-solid culture medium containing antibiotic selection pressure by adopting a filter membrane or liquid incubation method in vitro for dilution, coating and counting, the whole process is simple and convenient to operate, but consumes a long time and cannot be visualized, so that the migration risk of the drug-resistant genes under different water quality environmental conditions cannot be rapidly analyzed.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention constructs the escherichia coli carrying drug resistance and having double-color fluorescence, performs mixed culture on the escherichia coli, bacteria sensitive to various antibiotics and a water sample to be detected through the double-color fluorescence, performs bacterial fluorescence signal determination through a multi-dimensional panoramic flow cytometer, and calculates the horizontal migration frequency of the ARG according to the bacterial quantity results determined by different fluorescence signals. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided a method for assessing risk of migration of an antibiotic resistance gene, comprising:
(1) Providing a first target strain and a second target strain, wherein the first target strain has a bifluorescent signal and the second target strain has a multiple antibiotic susceptibility;
(2) Contacting the first target strain with the second target strain in a test sample under conditions suitable for conjugation to obtain a zygote;
(3) Performing fluorescence detection on the zygotes.
According to the method for evaluating the migration risk of the antibiotic resistance gene, the first target strain is preferably constructed by combining a red fluorescent protein-labeled recipient bacterium and a green fluorescent protein-labeled donor bacterium.
According to the method for evaluating the migration risk of the antibiotic resistance gene, the donor bacterium and the recipient bacterium are preferably Escherichia coli.
According to the method for evaluating the migration risk of the antibiotic-resistant gene, preferably, the escherichia coli comprises escherichia coli K12 or escherichia coli DH5 α.
According to the method for evaluating the migration risk of the antibiotic-resistant gene, the donor bacterium preferably has antibiotic resistance or carries a drug-resistant gene.
The method for evaluating the migration risk of antibiotic resistance genes, preferably, the antibiotic comprises at least one of tetracycline, ampicillin and kanamycin or a combination thereof; the drug resistance gene comprises: at least one of tetA, tnPR, and aphA, or a combination thereof.
According to the method for evaluating the risk of migration of an antibiotic-resistant gene of the present invention, preferably, the frequency of horizontal migration of the antibiotic-resistant gene = (green fluorescent signal channel bacterial detection amount-green fluorescent signal channel: red fluorescent signal channel bacterial detection amount)/(total bacterial amount-red fluorescent signal channel bacterial detection amount).
According to the method for evaluating the migration risk of the antibiotic resistance gene, the conditions suitable for jointing preferably refer to the temperature of 34-39 ℃, the speed of 150-200 rpm and the time of 5-12 h.
According to the method for evaluating the migration risk of the antibiotic resistance gene, the fluorescence signal detection is preferably performed by using a flow cytometer.
In a second aspect of the present invention, there is provided a system for assessing risk of migration of antibiotic-resistant genes, comprising:
a first target strain having a dual fluorescent signal;
a second target strain, wherein the second target strain is for gene transfer of E.coli, and the second target strain has multi-antibiotic sensitivity; and
provided is a multi-channel fluorescence detection device.
The present invention also provides the use of an agent for the manufacture of a kit for assessing the risk of migration of an antibiotic-resistant gene by a method comprising: (1) Providing a first target strain and a second target strain, wherein the first target strain has a dual fluorescent signal and the second target strain has a multi-antibiotic susceptibility; (2) Contacting the first target strain with the second target strain in a test sample under conditions suitable for conjugation to obtain a zygote; (3) performing fluorescence detection on the zygote;
the reagent comprises: a first target strain having a dual fluorescent signal;
a second target strain, wherein the second target strain is for gene transfer of E.coli, and the second target strain has multi-antibiotic sensitivity; and
a culture solution for culturing said first strain or said second strain, said culture solution comprising 0.01-0.5mM phosphate buffer, preferably 0.1 mM phosphate buffer.
The technical effects of the invention include but are not limited to:
(1) The method can quickly evaluate the risk of antibiotic drug-resistant gene migration under various water quality conditions (pH, conductivity, COD, antibiotics and other organic substances) of the water sample to be tested, effectively overcomes the defects of time and labor waste in the existing migration frequency calculation method, and can quickly evaluate the risk of drug-resistant gene horizontal migration under different environmental conditions.
(2) The method utilizes bacteria carrying bifluorescence and sensitive bacteria to detect the migration risk of antibiotic drug resistance genes in a water sample to be detected, the bacteria carrying bifluorescence is constructed to be donor bacteria, the sensitive bacteria is acceptor bacteria, the acceptor and the donor are jointed in the water sample to be detected, a large amount of bacteria are collected and fluorescence signals are detected to rapidly quantify the joint molecules, the horizontal migration risk of the ARG under various water quality conditions to be detected can be rapidly evaluated, and the method can be applied to the field of sewage risk evaluation.
(3) The fluorescence expression of the bifluorescence drug-resistant escherichia coli constructed by the invention has stability, the expression of green fluorescence cannot be lost due to the migration of plasmids, and an inducer is not needed for inducing the fluorescence expression; the screened aeromonas hydrophila has higher sensitivity to the drug resistance gene; the multidimensional panoramic flow cytometer has higher response signals to red fluorescence and green fluorescence, and can quickly evaluate the risk of antibiotic drug-resistant gene migration under the water quality conditions of different water samples to be tested.
(4) The bifluorescence drug-resistant escherichia coli constructed by the invention is used as a donor bacterium for researching horizontal joint transfer of antibiotic drug-resistant genes, can be applied to quickly calculating the horizontal joint frequency of the drug-resistant genes in a water body environment, and can also be used for quickly calculating the horizontal joint frequency of the drug-resistant genes in soil and aquatic organism intestinal tracts.
(5) The bifluorescence drug-resistant escherichia coli constructed by the invention is used as a donor bacterium for researching horizontal joint transfer of antibiotic drug-resistant genes, and even the drug-resistant genes can be rapidly positioned in intestinal tracts of plants or aquatic organisms through fluorescence signals, so that the drug-resistant gene migration visualization is realized.
Drawings
FIG. 1 is a photograph showing the colony growth morphology of two-color Escherichia coli constructed in the example of the present invention.
FIG. 2 is a scanning image of two-color Escherichia coli under a confocal scanning laser microscope constructed according to an embodiment of the present invention.
FIG. 3 is a flow cytogram of a water sample to be tested after co-culturing with bicolor Escherichia coli and sensitive Aeromonas hydrophila added.
FIG. 4 is a graph showing the calculated conjugation frequency of antibiotic resistance genes of different water samples to be tested according to the embodiment of the present invention.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated 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 in 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 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 herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
The method for evaluating the risk of migration of antibiotic-resistant genes of the present invention comprises the steps (1) to (3), which are described in detail below.
Step (1)
The first target strain of the present invention has a dual fluorescence signal, which means that it has two different detectable fluorescences, preferably red and green. The double-fluorescence signal strain is obtained by jointing an acceptor bacterium with red fluorescence protein and a donor bacterium with green fluorescence protein. The conjugation method is not particularly limited, and conjugation methods known in the art can be employed, and examples thereof include, but are not limited to, solid plate, liquid stationary culture, liquid shake culture (filtrate conjugation method), and the like.
Preferably, escherichia coli K12 carrying the red fluorescent protein in its genome is selected as the recipient bacterium, and Escherichia coli DH 5. Alpha. Carrying the green fluorescent protein is selected as the donor bacterium. The gene expressing the fluorescent protein may be carried by the genome of the host bacterium or operably linked to the gene via an expression vector. In certain embodiments, the genome of the recipient bacterium carries a gene that expresses a red fluorescent protein. In another embodiment, the donor bacterium contains a plasmid carrying a green fluorescent protein expression gene. The type of plasmid is not particularly limited, and preferably, the plasmid used in the present invention is an RP4 plasmid, which has a wide host range and high mobility.
Preferably, fluorescent bacterium K12 is labeled with mCherry red fluorescent protein and fluorescent bacterium DH5 α is labeled with sfgfp green fluorescent protein.
The terms "operably linked" and "label" as used herein have the meaning commonly understood in the art.
In the present invention, the donor bacterium in the first target strain is resistant to at least two antibiotics selected from the group consisting of tetracycline, ampicillin and kanamycin, and is, for example, resistant to all three antibiotics. Preferably, the donor bacteria in the first target strain carry at least two drug resistance genes selected from tetA, tnpR, aphA, for example, may simultaneously carry the above three drug resistance genes.
In the present invention, the donor bacterium: the recipient bacterium: LB medium =1:1-2: the bonding is carried out in a proportion of 1-5, and the bonding time is 5-12 h. Preferably, the donor bacterium: the recipient bacterium: LB medium =1:1-1.5:2-4, and the jointing time is 8-12h. Also preferably, the donor bacterium: the recipient bacterium: LB medium =1:1:3, the bonding time is 12h. The invention has the advantages that the jointing time is not more than 12h, and the jointing experiment time is greatly shortened.
Preferably, the temperature for culturing the donor bacterium, the recipient bacterium and the joining liquid is 34-41 ℃, preferably 35-39 ℃ and more preferably 36-38 ℃. The culture conditions for conjugation are 140rpm to 220rpm, preferably 150 rpm to 210 rpm, and more preferably 160 rpm to 200 rpm.
In the invention, after pure culture, washing, re-suspending and diluting are carried out until the number of the donor bacteria and the number of the recipient bacteria respectively reach 108-10 CFU/mL, preferably 109-10 CFU/mL, and further preferably 1010 CFU/mL.
It should be noted that, in the process of conjugating the donor bacterium and the recipient bacterium, neither red fluorescence nor green fluorescence needs to be added with an inducer for induced luminescence, the fluorescence expression of the constructed dual-fluorescence drug-resistant escherichia coli has stability, and the expression of green fluorescence cannot be lost due to the migration of plasmids.
Preferably, the green fluorescent protein (sfgfp) is measured using 488 nm excitation wavelength and the red fluorescent protein (mCherry) is measured using 581nm excitation wavelength. The fluorescence intensity measuring instrument is not particularly limited, and an apparatus or equipment known in the art may be employed, examples of which include, but are not limited to, for example, a confocal laser scanning microscope.
The second target strain of the present invention has multiple antibiotic sensitivity. Antibiotic sensitivity can be identified by drug sensitive tablets, examples of which include, but are not limited to: terramycin, roxithromycin, sulfanilamide, kanamycin, ampicillin and amoxicillin.
In the present invention, the source of the second target strain is not particularly limited, and it may be any antibiotic-sensitive bacteria from any sample. For example, the bacteria may be indigenous flora derived from water or 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 (e.g., municipal 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 isolated from a mixture of excreta of rana tropicalis by culturing, which has multiple antibiotic sensitivities and can undergo gene transfer with escherichia coli.
Step (2)
In the step (2), the first target strain is contacted with the second target strain in a sample to be tested under the condition suitable for conjugation to obtain a conjugant.
Preferably, the sample to be tested is subjected to filter sterilization treatment. The filter sterilization step is not particularly limited, and for example, the sample to be tested may be sterilized by passing through a filter having a specific pore size, usually 0.22 μm or less.
The conditions suitable for conjugation mean that the temperature of the mixed culture is 34 to 41 ℃, preferably 35 to 39 ℃, and more preferably 36 to 38 ℃. The culture conditions for conjugation are from 140rpm to 220rpm, preferably from 150 rpm to 210 rpm, and more preferably from 160 rpm to 200rpm, of a constant temperature shaker. The bonding time is 8-15 h. Still more preferably 8-12h.
Preferably, the bacterial amounts of the first and second target strains are preset to 109, 1010, 1011 CFU/mL.
The sample to be tested in the 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 is understood that the sample to be tested may be derived from any plant or animal sample of the above-mentioned body of water or soil.
Step (3)
And (3) performing flow cytometer fluorescence quantification by adopting laser with a specific wavelength. The type of flow cytometer is not particularly limited as long as it has multiple fluorescence channels.
According to the invention, the migration frequency of the drug-resistant gene in the sample to be detected can be calculated according to the bacterial quantity results measured by different fluorescence channels of the flow cytometer. The flow cytometer is adopted to identify fluorescence, so that accurate identification, counting and sorting can be carried out according to different fluorescence colors of a donor, a receptor and a conjugant.
Preferably, when the fluorescence result is measured in step (3), a gradient dilution is performed to collect the fluorescence result under the condition of selecting the optimal concentration, for example, the pretreatment of the computer-loaded sample is performed according to the absorbance at OD600nm, and the preset range is 0.5-0.7. More preferably, in the step (3), the single fluorescence interference is eliminated by measuring the Escherichia coli K12 fluorescence signal and the Escherichia coli DH5 alpha fluorescence signal, the amount of the collected bacteria is preset to be 5000-10000, and the collected bacteria are distributed in a fluorescence scattering point manner on a coordinate axis.
In step (3) of the present invention, the conjugation frequency is calculated from the ratio of fluorescence signals collected from different channels, preferably, the frequency of horizontal migration of antibiotic resistance gene = (green fluorescence signal channel bacterial detection amount-green fluorescence signal channel: red fluorescence signal channel bacterial detection amount)/(total amount of bacteria-red fluorescence signal channel bacterial detection amount). In a specific embodiment, ch02 channel collects bacterial amount of sfgfp fluorescent protein positive signal, ch06 channel collects bacterial amount of mCherry fluorescent protein positive channel, bifluorescent bacteria simultaneously generate fluorescent signal at Ch02 and Ch06 channels, and zygote generates fluorescent signal at Ch02 channel, and frequency of horizontal migration of ARG = (Ch 02 bacterial detection amount-Ch 02: ch06 bacterial detection amount)/(bacterial total amount-Ch 06 bacterial detection amount).
In the invention, the jointing liquid obtained in the step (2) can be directly counted by a flow cytometer without multiple times of culture, the initial growth condition of the joint in the original jointing environment is ensured, the number of partial joints which can not be cultured can be effectively collected, and the accuracy of jointing frequency is improved. In addition, the zygomorph comprises not only a part of flora which can be cultured, but also a part of flora which can not be cultured, the calculated conjugation frequency is more accurate, and the experimental error brought by multiple times of culture is reduced.
It will be appreciated by those skilled in the art that additional steps or operations may be included before or after steps (1) - (3) above, or between any of these steps, for example to further optimize and/or improve the methods of the present invention.
The bifluorescence drug-resistant escherichia coli constructed by the invention is used as a donor bacterium for researching horizontal joint transfer of antibiotic drug-resistant genes, and even the drug-resistant genes can enter the intestinal tracts of plants or aquatic organisms to be quickly positioned through fluorescence signals, so that the drug-resistant gene migration visualization is realized.
Examples
This example is a method for evaluating the risk of migration of antibiotic-resistant genes, and is described below.
(1) Taking out the red fluorescence escherichia coli K12 and the green fluorescence escherichia coli DH5 alpha from a refrigerator at the temperature of-80 ℃, coating after activating, and checking whether the fluorescence proteins of the red fluorescence escherichia coli and the green fluorescence escherichia coli are normally expressed.
(2) Each of the two E.coli strains was selected and cultured in LB medium containing 100. Mu.g/mL ampicillin, 50. Mu.g/mL kanamycin and 10. Mu.g/mL tetracycline hydrochloride to logarithmic growth phase in which E.coli DH 5. Alpha. Was cultured.
(3) Washing with physiological saline or sterile phosphate buffer (0.1 mM) and diluting Escherichia coli K12 and Escherichia coli DH5 alpha until the bacterial count reaches 1010 CFU/mL respectively to obtain the pre-joined bacterial liquid.
(4) Taking red escherichia coli K12 as a recipient bacterium, taking green escherichia coli DH5 alpha as a donor bacterium, and carrying out the following steps: recipient bacterium: LB medium =1:1: the bonding is carried out in a ratio of 3, the overall bonding is designed to be 20 mL, and the bonding time is 12h.
(5) The joining liquid is taken to be coated on an LB solid medium, after 18 h is cultured, the fluorescence signal of each single colony is checked by an inverted fluorescence microscope, and colonies with double fluorescence signals are selected.
(6) And (3) performing secondary LB liquid amplification culture on the selected double-fluorescence signal bacterial colonies, respectively sucking 2 mL bacterial liquid to a 24-hole cell culture plate, and observing fluorescence signals by using an inverted fluorescence microscope.
(7) The bacterial fluid in which the red and green fluorescence signals are stable wells was selected and streaked to isolate single colonies as shown in FIG. 1.
(8) The single colony separated is observed by fluorescence signals through an inverted fluorescence microscope, and is repeated for a plurality of times to screen out bicolor fluorescence escherichia coli with the best fluorescence signals and the best growth conditions as target strains, as shown in figure 2, the bicolor fluorescence escherichia coli in figure 2 shows that stable fluorescence signals appear under excitation wavelengths of 488 nm and 581 nm.
(9) Sucking 10 mL of the liquid manure mixture from a tropical Rana unguiculata breeding cylinder, centrifuging at a high speed, gently sucking the supernatant, sucking 1 mL precipitate into LB culture medium by a liquid-moving machine, and culturing 24 h at 37 ℃ and 180 rpm.
(10) After the bacterial liquid is diluted in a gradient manner, 100 mu L of the bacterial liquid is coated in LB solid culture medium and cultured at 37 ℃ for 24 h.
(11) Selecting single bacterial colonies with different bacterial colony morphology characteristics in a culture dish, carrying out LB liquid expansion culture, detecting the sensitivity of different bacterial liquids by using drug sensitive tablets of oxytetracycline, roxithromycin, sulfanilamide, kanamycin, ampicillin and amoxicillin, and finally selecting bacteria sensitive to various antibiotics for strain identification to determine the bacteria as aeromonas hydrophila.
(12) Washing with physiological saline, adjusting overnight cultured bicolor fluorescent bacteria (donor bacteria) and aeromonas hydrophila (recipient bacteria), centrifuging at 8000Xg for 6 min, collecting precipitate, and resuspending the collected bacteria with LB culture medium to reach 1010 CFU/mL.
(13) Taking Zhujiang water, artificial lake water, lake water and disinfected effluent as water samples to be detected, taking deionized water as blank control, and performing membrane sterilization treatment on the water samples to be detected through a 0.22-micron organic filter membrane to obtain membrane water samples to be detected.
(14) The well-adjusted bicolor fluorescent bacteria (donor bacteria) and aeromonas hydrophila (recipient bacteria) are respectively taken and added with 4 mL into 17 mL of each to-be-detected transmembrane water sample (containing 5 mL of LB culture solution) to be cultured in a constant temperature shaking table at 37 +/-2 ℃ and 180 rpm for 12h to obtain the joint solution.
(15) The above-mentioned binding solution 8 mL was centrifuged at 8000xg for 8 min in a centrifuge, the filtrate was discarded, and the bacterial solution was washed repeatedly with 8 mL phosphate buffer (0.1 mM) for 3 times, and then adjusted to OD600 nm. Apprxeq.0.7.
(16) After the pretreated junction fluid is screened by a 70-micron sterile cell, 1.5 mL centrifuge tube is used for collecting 1 mL bacterial fluid, a flow cytometer is used for collecting fluorescence signals, the total quantity of collected cells is set to 10000, 488 nm excitation light wavelength is used for collecting bacterial quantity of green fluorescence protein (sfgfp) signals in a Ch02 channel, and 581nm excitation light wavelength is used for collecting bacterial quantity of red fluorescence protein (mCherry) signals in a Ch06 channel.
The flow cytometry imaging result after adding bicolor escherichia coli and sensitive aeromonas hydrophila to a water sample to be detected for co-culture is shown in fig. 3, the aeromonas hydrophila is taken as a receptor bacterium and does not generate fluorescence signals in Ch02 and Ch06 channels, and the bicolor fluorescence bacterium is taken as a donor bacterium and respectively generates obvious green and red fluorescence signals in the Ch02 and Ch06 channels; after the bacteria are co-cultured, the bicolor fluorescent bacteria lose green fluorescent signals in a Ch02 channel due to the migration of plasmids, only an independent red fluorescent signal appears in the Ch06 channel, and the Aeromonas hydrophila obtains the independent green fluorescent signals appearing in the Ch02 channel of the plasmids, namely, the zygote.
(17) The frequency of horizontal migration of ARG = (Ch 02 bacteria detection amount-Ch 02: ch06 bacteria detection amount)/(total bacteria amount-Ch 06 bacteria detection amount), and the migration risk calculation results are plotted in fig. 4. FIG. 4 shows that the migration frequency of the resistance gene is highest in deionized water compared with other surface water and disinfected effluent; the disinfected effluent also exhibits a higher junction frequency compared with surface water, and the resistance gene migration frequency of the artificial lake water in the surface water is the lowest and meets the expectation. And the migration frequency detection difference is obvious according to the difference of the comprehensive water quality conditions, which shows that the method can rapidly evaluate the risk of the migration of antibiotic drug resistance genes under the water quality conditions (pH, conductivity, COD, antibiotics and other organic substances) of various water samples to be detected.
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. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass 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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