阅读说明：本技术 一种评估抗性基因迁移风险的方法 (Method for evaluating migration risk of resistance gene ) 是由 许燕滨 欧阳朋倩 谢光炎 罗宏威 陈卓瑶 李宇 于 2021-01-18 设计创作，主要内容包括：本申请属于生物技术的领域,尤其涉及一种评估抗性基因迁移风险的方法。本申请提供了一种评估抗性基因迁移风险的方法,以荧光细菌为受体,耐药细菌为供体,利用荧光细菌和耐药细菌检测某水质下抗性基因迁移风险的一种方法,通过构建荧光细菌的荧光强度F-吸光度-荧光细菌浓度C三者标准曲线；根据标准曲线得到在待测水样中接合后受体的浓度,以及筛选液中接合子的浓度,实现检测待测水样中抗性基因迁移频率的目的。本申请提供了一种评估抗性基因迁移风险的方法,能有效解决现有计算迁移频率的方法中存在的费时费力,以及无法评估水样中重金属、抗生素及其他有机物质对抗性基因迁移综合风险的技术缺陷。(The application belongs to the field of biotechnology, and particularly relates to a method for evaluating migration risk of a resistance gene. The application provides a method for evaluating migration risk of a resistance gene, which is a method for detecting migration risk of the resistance gene under certain water quality by using fluorescent bacteria as an acceptor and drug-resistant bacteria as a donor and by constructing a standard curve of fluorescence intensity F-absorbance-fluorescence bacteria concentration C of the fluorescent bacteria; and obtaining the concentration of the receptor after the conjugation in the water sample to be detected and the concentration of the zygote in the screening solution according to the standard curve, thereby realizing the purpose of detecting the migration frequency of the resistant gene in the water sample to be detected. The application provides a method for evaluating migration risk of a resistance gene, which can effectively overcome the technical defects that the existing method for calculating migration frequency wastes time and labor, and the comprehensive risk of heavy metals, antibiotics and other organic matters in a water sample to the migration of the resistance gene cannot be evaluated.)

1. A method of assessing the risk of migration of a resistance gene, comprising:

step 1, diluting fluorescent bacteria into a dilution solution with a series of concentration gradients with absorbance in a preset range, calculating the fluorescent bacteria concentration of the dilution solution, and constructing a standard curve of absorbance-fluorescent bacteria concentration C;

diluting the fluorescent bacteria into a dilution solution with a series of concentration gradients of which the absorbance is in a preset range, measuring the fluorescence intensity of the dilution solution, and constructing a standard curve of absorbance-fluorescence intensity F;

wherein the fluorescent bacterium has a fluorescent protein gene and a first antibiotic resistance gene, so that the fluorescent bacterium is resistant to the first antibiotic, fluoresces under induction of an inducer and preset excitation light, and is sensitive to a second antibiotic;

step 2, mixing and culturing the fluorescent bacteria, the drug-resistant bacteria, the water sample to be detected and the inducer to obtain a first mixture, so that the absorbance of the fluorescent bacteria and the drug-resistant bacteria in the first mixture is within the preset range, and the absorbance of the fluorescent bacteria and the drug-resistant bacteria are equal; the drug-resistant bacteria have resistance to the second antibiotic, the water sample to be detected is subjected to filtration sterilization treatment in advance, and the inducer is a substance for inducing the expression of the fluorescent protein;

the method comprises the following steps of (1) performing mixed culture on the fluorescent bacteria and the drug-resistant bacteria to obtain first fluorescent bacteria; measuring the fluorescence intensity of the first mixture under the preset excitation light, and calculating the concentration of the first fluorescent bacteria in the first mixture according to the standard curve of the absorbance-fluorescence intensity F and the standard curve of the absorbance-fluorescent bacteria concentration C, wherein the concentration of the first fluorescent bacteria is the concentration of the acceptor;

wherein a second mixture is obtained after the first mixture, the first antibiotic and the second antibiotic are mixed and cultured; after the fluorescent bacteria are jointed with the drug-resistant bacteria, second fluorescent bacteria are obtained; measuring the fluorescence intensity of the second mixture under the preset excitation light, and calculating the concentration of the second fluorescent bacteria in the second mixture according to the standard curve of the absorbance-fluorescence intensity F and the standard curve of the absorbance-fluorescent bacteria concentration C, wherein the concentration of the second fluorescent bacteria is the concentration of the zygote;

and 3, calculating the migration frequency of the resistance genes in the water sample to be detected according to the concentration of the receptor and the concentration of the zygote.

2. The method of claim 1, wherein the fluorescent protein gene is selected from one of green fluorescent protein, red fluorescent protein, blue fluorescent protein, and yellow fluorescent protein.

3. The method according to any one of claims 1 to 2, wherein the first antibiotic resistance gene is an aminoglycoside resistance gene; the second antibiotic is an antibiotic other than the first antibiotic.

4. The method according to any one of claims 1 to 3, wherein the fluorescent bacterium is selected from the group consisting of EGFP-BL 21; the drug-resistant bacterium is one of bacteria which are drug-resistant bacteria in the environment and can perform gene migration with escherichia coli.

5. The method of claim 4, wherein the fluorescent bacterium is EGFP-BL21 and the inducing agent is IPTG, isopropyl- β -D-thiogalactoside.

6. The method according to any one of claims 1 to 5, wherein in step 1, the absorbance is OD600 nm; the preset range is 0.1-0.9.

7. The method according to any one of claims 1 to 5, wherein in the step 2, the absorbance of the fluorescent bacteria in the water sample to be detected is 0.1; and the absorbance of the drug-resistant bacteria in the water sample to be detected is 0.1.

8. The method as claimed in claim 1, wherein in step 2, the filter sterilization treatment comprises sterilizing the water sample to be tested through a 0.22 μm organic filter membrane.

9. The method according to any one of claims 1 to 8, wherein in step 2, the temperature of the mixed culture is 30 ℃ ± 2 ℃; the mixed culture time is 16-24 h; the mixed culture condition is a constant temperature shaking table at 150 rpm-200 rpm.

10. The method according to any one of claims 1 to 9, wherein in step 2, the frequency of migration of the resistance gene in the test water sample is the concentration of the zygote/the concentration of the receptor.

Technical Field

The application belongs to the field of biotechnology, and particularly relates to a method for evaluating migration risk of a resistance gene.

Background

With the wide application of antibiotics, the follow-up effect brought by the antibiotics gradually attracts people's attention, and the most important one is the development of drug-resistant bacteria caused by the antibiotics. The conventional risk evaluation method is usually a risk entropy accumulation method, namely, a risk entropy is calculated according to the residual concentration of antibiotics detected in water and the risk entropy of various antibiotics is accumulated, but the actual environmental water sample usually has complex components, for example, the aquaculture wastewater may contain various hormones, and the industrial wastewater may contain various heavy metals, and the existing research has proved that the existence of partial organic matters and heavy metals can promote the migration of resistance genes and the development of drug-resistant bacteria. Therefore, the influence of organic matters and heavy metals in water on the process of acquiring drug resistance of bacteria is not considered when the risk evaluation of antibiotics in water is carried out by only a risk entropy accumulation method. Moreover, the method for calculating the migration frequency usually uses dilution plate coating, the process usually takes a long time, and the experiment failure caused by the error of concentration control is easy to occur, and the number of samples is not too large, so that the multi-sample test cannot be performed quickly.

Disclosure of Invention

In view of the above, the present application provides a method for evaluating migration risk of resistance genes, which can effectively solve the technical defects that the existing method for calculating migration frequency wastes time and labor, and the migration risk of heavy metals, antibiotics and other organic substances in a water sample to the resistance genes cannot be evaluated.

The application provides a method for evaluating migration risk of a resistance gene, which comprises the following steps:

step 1, diluting fluorescent bacteria into a dilution solution with a series of concentration gradients with absorbance in a preset range, calculating the fluorescent bacteria concentration of the dilution solution, and constructing a standard curve of absorbance-fluorescent bacteria concentration C;

diluting the fluorescent bacteria into a dilution solution with a series of concentration gradients of which the absorbance is in a preset range, measuring the fluorescence intensity of the dilution solution, and constructing a standard curve of absorbance-fluorescence intensity F;

wherein the fluorescent bacterium has a fluorescent protein gene and a first antibiotic resistance gene, so that the fluorescent bacterium is resistant to the first antibiotic, fluoresces under induction of an inducer and preset excitation light, and is sensitive to a second antibiotic;

step 2, mixing and culturing the fluorescent bacteria, the drug-resistant bacteria, the water sample to be detected and the inducer to obtain a first mixture, so that the absorbance of the fluorescent bacteria and the drug-resistant bacteria in the first mixture is within the preset range, and the absorbance of the fluorescent bacteria and the drug-resistant bacteria are equal; the drug-resistant bacteria have resistance to the second antibiotic, the water sample to be detected is subjected to filtration sterilization treatment in advance, and the inducer is a substance for inducing the expression of the fluorescent protein;

the method comprises the following steps of (1) performing mixed culture on the fluorescent bacteria and the drug-resistant bacteria to obtain first fluorescent bacteria; measuring the fluorescence intensity of the first mixture under the preset excitation light, and calculating the concentration of the first fluorescent bacteria in the first mixture according to the standard curve of the absorbance-fluorescence intensity F and the standard curve of the absorbance-fluorescent bacteria concentration C, wherein the concentration of the first fluorescent bacteria is the concentration of the acceptor;

after the first mixture, an inducer, a first antibiotic and a second antibiotic are mixed and cultured, a second mixture is obtained, the fluorescent bacteria are jointed with the drug-resistant bacteria, second fluorescent bacteria are obtained, the fluorescence intensity of the second mixture is measured under the preset exciting light, and the concentration of the second fluorescent bacteria in the second mixture is calculated according to the standard curve of the absorbance-fluorescence intensity F and the standard curve of the absorbance-fluorescence bacteria concentration C, wherein the concentration of the second fluorescent bacteria is the concentration of a joint compound;

and 3, calculating the migration frequency of the resistance genes in the water sample to be detected according to the concentration of the receptor and the concentration of the zygote.

In other embodiments, the fluorescent protein gene is selected from one of green fluorescent protein, red fluorescent protein, blue fluorescent protein, or yellow fluorescent protein.

Specifically, the fluorescence intensity of the diluent is measured by using the wavelength of the excitation light of the fluorescent protein. For example, green fluorescent protein (EGFP) uses 488nm excitation wavelength and fluorescence intensity is collected by confocal laser scanning microscope.

In other embodiments, the first antibiotic resistance gene is selected from the group consisting of aminoglycoside resistance genes; the second antibiotic is an antibiotic other than the first antibiotic.

In other embodiments, the first antibiotic resistance gene is selected from one of aminoglycoside resistance genes; the second antibiotic is an antibiotic other than the first antibiotic.

Specifically, the second antibiotic is selected from beta-lactamase antibiotics, tetracycline antibiotics and quinolone antibiotics.

Specifically, the aminoglycoside resistance gene is a kanamycin resistance gene; the beta-lactamase antibiotic is amoxicillin.

In other embodiments, the drug-resistant bacterium is one of bacteria that are environmentally resistant and that can undergo gene transfer with e.

In other embodiments, the resistant bacteria is selected from resistant shigella.

In other embodiments, the fluorescent bacterium is EGFP-BL21 and the inducing agent is isopropyl- β -D-thiogalactoside IPTG.

In other examples, the green fluorescent E.coli was obtained by recombination of a plasmid (pET28a) containing green fluorescent protein (EGFP) and kanamycin resistance gene, which was introduced into E.coli (BL 21).

In other embodiments, in step 1, the absorbance is OD600 nm; the preset range is 0.1-0.9.

In other embodiments, in step 2, the absorbance of the fluorescent bacteria in the water sample to be detected is 0.1; and the absorbance of the drug-resistant bacteria in the water sample to be detected is 0.1.

In other embodiments, in step 2, the filter sterilization treatment comprises sterilizing the water sample to be tested through a 0.22 μm organic filter membrane.

In other embodiments, in step 2, the temperature of the mixed culture is 30 ℃ ± 2 ℃; the mixed culture time is 16-24 h; the mixed culture condition is a constant temperature shaking table at 150 rpm-200 rpm.

In other embodiments, in step 2, the time for the mixed culture is 24 hours.

In other embodiments, in step 2, the frequency of migration of the resistance gene in the test water sample is the concentration of the zygote/the concentration of the receptor.

Specifically, in step 1, the method for calculating the fluorescent bacteria concentration of the diluent is a conventional dilution coating calculation method, and the bacteria concentration of each gradient is calculated according to a dilution coating counting method.

Specifically, in step 1, the method for measuring the fluorescence intensity of the diluent is to use the conventional confocal laser scanning microscope to collect the fluorescence intensity of the diluent.

Specifically, in step 2, when the concentration of the connexon and the concentration of the receptor are measured, if the fluorescence intensity of the measured sample is too strong, a proper amount of dilution is needed, for example, after pre-balancing according to the use method of a 24-well culture well plate of a laser confocal scanning microscope, 500 μ L of bacterial liquid is sucked and added to the 24-well culture well plate containing 2mL of nutrient broth culture liquid in each well (namely, diluted by 5 times), and the laser confocal scanning microscope acquires a fluorescence image to obtain an average fluorescence intensity value; if the fluorescence intensity of the measured sample is proper, pre-balancing according to a use method of a 24-hole culture pore plate of a laser confocal scanning microscope, directly absorbing 2mL of bacterial liquid, adding the bacterial liquid into the 24-hole culture pore plate, and directly collecting a fluorescence image by using the laser confocal scanning microscope to obtain an average fluorescence intensity value; if the fluorescence intensity of the sample is too weak, the experiment needs to be carried out again, and the concentrations of the fluorescent bacteria and the drug-resistant bacteria in the first mixture are increased.

The application relates to a method for detecting migration risk of resistance genes in a water sample to be detected by using fluorescent bacteria and drug-resistant bacteria. Constructing a standard curve of fluorescence intensity F-absorbance-fluorescence bacterium concentration C of the fluorescence bacterium by using the fluorescence bacterium as an acceptor and using the drug-resistant bacterium as a donor; the method comprises the following steps of (1) carrying out mixed culture on fluorescent bacteria, drug-resistant bacteria, a water sample to be detected and an inducer to obtain a first mixture, wherein the inducer induces the expression of fluorescent protein of the fluorescent bacteria, and the fluorescence intensity of the first fluorescent bacteria, namely an acceptor, in the first mixture is detected after the acceptor is jointed with a donor; the first mixture, the first antibiotic and the second antibiotic are mixed and cultured to obtain a second mixture, the drug-resistant bacteria have a second antibiotic resistance gene due to the resistance of the drug-resistant bacteria to the second antibiotic, the drug-resistant bacteria serve as donor bacteria and can migrate the drug-resistant gene (namely the second antibiotic resistance gene) into the fluorescent bacteria in a water sample to be detected, and the second fluorescent bacteria after the fluorescent bacteria are jointed with the drug-resistant bacteria are connexons, so that the connexons have the second antibiotic resistance gene; meanwhile, the second antibiotic kills recipient bacteria that do not obtain the second antibiotic resistance gene, the zygomotor is fluorescent bacteria containing the second antibiotic resistance gene, so that the zygomotor bacteria are resistant to the first antibiotic and the second antibiotic, the inducer is used to induce the expression of the fluorescent protein, fluorescence is emitted in the second mixture, and the fluorescence intensity of the zygomotor bacteria in the second mixture is detected. The concentration of the first fluorescent bacteria, namely the receptor, in the first mixture and the concentration of the second fluorescent bacteria, namely the zygote, in the second mixture are obtained according to the standard curve, and the aim of detecting the migration frequency of the resistance genes in the water sample to be detected can be fulfilled by comparing the concentrations of the first fluorescent bacteria, namely the receptor, and the second fluorescent bacteria, namely the zygote, in the second mixture, the migration frequency of the resistance genes in the water sample to be detected can be quickly determined, so that the method can be applied to the.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a fluorescence intensity F-OD600nm absorbance standard curve provided in the examples herein;

FIG. 2 is a scanning image of a confocal scanning laser microscope at different dilution concentrations of EGFP-BL21 of green fluorescent Escherichia coli provided in the examples of the present application;

FIG. 3 is a scanning image of a confocal scanning laser microscope after co-culture for 24 hours by adding EGFP-BL21, drug-resistant Shigella and IPTG to different water samples to be tested, which is provided by the embodiment of the application;

FIG. 4 is a scanning image of a confocal scanning laser microscope after co-culture for 24 hours by adding green fluorescent Escherichia coli EGFP-BL21, drug-resistant Shigella, IPTG, kanamycin and amoxicillin to different water samples to be detected, which is provided by the embodiment of the application;

FIG. 5 shows the migration frequency of resistance genes of different water samples to be tested according to the present invention.

Detailed Description

The application provides a method for evaluating migration risk of resistance genes, which is used for solving the technical defects that time and labor are wasted and the migration risk of heavy metals, antibiotics and other organic matters in a water sample to the resistance genes cannot be evaluated in the prior art.

The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Wherein, the raw materials or reagents used in the following examples are all sold in the market or made by the user; the nutrient broth used in the following examples is an existing conventional bacterial culture medium.

The method comprises three parts of establishment of a relationship between fluorescence intensity F-OD600nm absorbance and bacterial concentration C, receptor coupling in a water sample to be detected and detection of fluorescence intensity by a laser confocal scanning microscope, and comprises the following steps:

taking out the green fluorescent escherichia coli from the refrigerator, coating the green fluorescent escherichia coli after activation, and checking whether the fluorescent protein of the green fluorescent escherichia coli is normally expressed or not.

Activated green fluorescent E.coli was diluted to a series of concentration-gradient dilutions with OD600nm in the range of 0.1-0.9.

And (3) taking the diluent for plate coating, recording the green fluorescence escherichia coli concentration corresponding to each OD600nm absorbance value, and fitting the data to obtain a standard curve of OD600nm absorbance-green fluorescence escherichia coli concentration C.

Taking 2mL of diluent to a 24-hole laser confocal scanning microscope plate pre-balanced for 30min by 3mL of phosphate buffer (0.1mM), and collecting a fluorescence image on a computer laser confocal scanning microscope, wherein a Zeiss ZEN system is adopted for image collection, 488nm excitation light (namely wavelength for EGFP) is selected for image collection, the gain value and the Effective NA value of a detector are adjusted according to the principle that the image is not exposed when the image is collected at the maximum concentration, the data is kept unchanged in the subsequent image collection, the concentration of green fluorescence escherichia coli in the diluent is determined according to the collected fluorescence image, and the data is fitted to obtain a fluorescence intensity F-OD600nm absorbance standard curve.

Collecting a water sample to be detected, and filtering the water sample by using a 0.22 mu m organic filter membrane for sterilization treatment.

Adjusting the concentration of donor bacteria and acceptor bacteria cultured overnight to be the same by using nutrient broth, and equivalently adding the donor bacteria and the acceptor bacteria into a water sample subjected to degerming treatment to obtain a first bacteria-containing water sample; wherein the recipient bacterium is a green fluorescent escherichia coli (the green fluorescent escherichia coli is sensitive to the second antibiotic) and the donor bacterium is a drug-resistant bacterium (the drug-resistant bacterium is resistant to the second antibiotic).

The first bacteria-containing water sample is mixed and cultured for 24 hours at the temperature of 30 +/-2 ℃ and the constant-temperature shaking table at 150rpm to obtain a second bacteria-containing water sample, and the concentration of the fluorescent bacteria in the second bacteria-containing water sample is the concentration of the receptor; in order to ensure that the fluorescence intensity is in a proper range, adding 2mL of phosphate buffer (0.1mM) into a culture well plate of a 24-well laser confocal scanning microscope for pre-balancing for 30min, then adding 500 mu L of a bacteria-containing water sample into the well plate added with 2mL of phosphate buffer, wherein the phosphate buffer contains an inducer (a substance capable of inducing green fluorescent protein expression), collecting a fluorescence image of a sample by using the laser confocal scanning microscope, obtaining the average fluorescence intensity according to the fluorescence image, obtaining the concentration of recipient bacteria by using the fluorescence intensity F-OD600nm absorbance-the concentration C of the bacteria, and multiplying the concentration by a dilution factor of 5 during subsequent data processing.

Meanwhile, adding a first water sample containing bacteria into nutrient broth containing a first antibiotic, a second antibiotic and an inducer, and carrying out shake culture at the constant temperature of 30 +/-2 ℃ and 150rpm for 24 hours to obtain a bacterial liquid as a zygospore bacterial screening liquid. After the fluorescent bacteria in the zygospore bacteria screening solution are jointed with the drug-resistant bacteria, second fluorescent bacteria are obtained, and the concentration of the second fluorescent bacteria is the concentration of the zygospore; in order to ensure that the fluorescence intensity is in a proper range, 2mL of phosphate buffer (0.1mM) is added into a culture pore plate of a 24-pore laser confocal scanning microscope for pre-balancing for 30min, 2mL of zygospore bacteria screening solution is added into the pore plate, a laser confocal scanning microscope is used for carrying out fluorescence image acquisition on a sample, the average fluorescence intensity is obtained according to the fluorescence image, and the concentration of the zygospore bacteria is obtained by utilizing the fluorescence intensity F-OD600nm absorbance-bacteria concentration C.

And (3) obtaining the migration frequency of the resistance genes under the water quality condition of the water sample to be detected according to a formula, namely the concentration of the zygospermobacter/the concentration of the receptor bacteria.

Specifically, when a confocal laser scanning microscope is used for collecting fluorescence images, the concentration of green fluorescence escherichia coli in liquid in a culture well plate of the confocal laser scanning microscope with 24 wells needs to be controlled to a certain extent, and when the green concentration of the green fluorescence escherichia coli is too high or too low, fluorescence intensity data may not be accurate enough. Therefore, the embodiment of the present application performs dilution processing when acquiring the fluorescence intensity of the recipient bacteria, and the fluorescence intensity data of the conjugator bacteria is acquired at the original concentration. Meanwhile, when a confocal laser microscope is used for collecting a fluorescence image, camera data needs to be adjusted from a sample with the maximum fluorescence intensity, otherwise, an overexposure phenomenon possibly occurs to influence the fluorescence intensity value, and therefore, the fluorescence intensity of the receptor or the conjugant bacteria needs to be diluted in time if the fluorescence intensity is too high.

Specifically, the drug-resistant bacteria can be selected from drug-resistant Shigella screened from natural environment. The application relates to a method for detecting migration risk of a resistance gene under certain water quality by using green fluorescent escherichia coli and shigella. Firstly, preparing fresh bacterial liquid of green fluorescent escherichia coli and shigella, wherein the green fluorescent escherichia coli has kanamycin resistance, and the shigella has resistance of a resistance gene to be detected (in the application, an amoxicillin is taken as a representative example for a beta-lactamase resistance gene). And drawing a fluorescent colibacillus fluorescence intensity F-OD600nm absorbance-bacterial concentration C standard curve. Adding equivalent fluorescent escherichia coli and shigella into a to-be-detected transmembrane water sample containing a fluorescent protein inducer for jointing, collecting a fluorescent image by using a laser confocal scanning microscope after 24 hours, obtaining fluorescence intensity data in the water sample containing bacteria, and obtaining the number of receptors according to a standard curve. Meanwhile, adding a water sample containing bacteria into nutrient broth (namely screening solution) containing kanamycin, amoxicillin and a fluorescent protein inducer, culturing for 24h in a constant-temperature shaking table at 30 +/-2 ℃ and 150rpm, measuring the fluorescence intensity in the screening solution by using a laser confocal scanning microscope, and obtaining the quantity of the zygotes according to a standard curve. The frequency of resistance gene migration in water samples is known as the number of zygotes/number of receptors.

Example 1

The embodiment of the application provides the standard curve drawing of the absorbance OD600 nm-fluorescence intensity F, and comprises the following processes:

the overnight cultured green fluorescent Escherichia coli EGFP-BL21 was taken out, diluted with the nutrient broth culture, and the absorbance at 600nm was measured with a spectrophotometer to be within the range of 0.1 to 0.9, with a dilution concentration gradient OD600nm of: 0. 0.062, 0.095, 0.208, 0.326, 0.430, 0.614.

2mL of phosphate buffer (0.1mM) was added to a 24-well confocal scanning laser microscopy culture well plate for pre-equilibration, and the mixture was poured out after 30 min.

2mL of the dilution was added to a pre-equilibrated 24-well confocal scanning laser microscopy culture well plate.

Collecting fluorescence image of culture well plate of confocal laser scanning microscope by on-board confocal laser scanning microscope, adjusting detector gain value to 650V and Effective NA value to 0.45 according to the above photographing principle, obtaining average fluorescence intensity data by obtaining concentration gradient picture as shown in figure 2, and drawing fluorescence intensity F-OD600nm absorbance standard curve (R)²0.9863), the results are shown in FIG. 1; in FIG. 2, 1 is a scanning picture of a confocal laser scanning microscope with a dilution concentration gradient of 0.062, 2 is a scanning picture of a confocal laser scanning microscope with a dilution concentration gradient of 0.095, and 3 is a scanning picture of a confocal laser scanning microscope with a dilution concentration gradient of 0.095The scanning picture of the confocal laser scanning microscope when the dilution concentration gradient is 0.208, 4, 5 and 6 are respectively the scanning picture of the confocal laser scanning microscope when the dilution concentration gradient is 0.326, 0.430 and 0.614, respectively.

Example 2

The application provides a resistance gene migration risk assessment test in typical process water quality of duck breeding wastewater, which comprises the following steps:

taking out the green fluorescence escherichia coli cultured overnight, diluting the green fluorescence escherichia coli with nutrient broth culture solution, measuring the absorbance at 600nm by using a spectrophotometer to enable the absorbance to be within the range of 0.1-0.9, calculating the concentration of the green fluorescence escherichia coli in the diluent by adopting a dilution coating counting method, and constructing a standard curve of the absorbance-the concentration C of the green fluorescence escherichia coli.

Taking the water inlet and outlet of a primary sedimentation tank, an adjusting tank, a primary A/O (anoxic/oxic) and a secondary A/O (anoxic/oxic) outlet water and a disinfection tank in a Guangdong certain duck wastewater treatment process as water samples to be detected, taking pure water as a blank control, and performing membrane-filtration degerming treatment on the water samples to be detected through a 0.22-micrometer organic filter membrane to prepare membrane-filtered water samples to be detected.

Taking overnight cultured green fluorescent escherichia coli EGFP-BL21 and drug-resistant Shigella (resistant to amoxicillin), adjusting the concentrations of the green fluorescent escherichia coli EGFP-BL21 and the drug-resistant Shigella to OD600nm to 0.6, adding 10mL of bacterial liquid into 40mL of membrane water samples containing IPTG (0.1mmol/mL) to be detected, namely the concentrations of two bacteria in water are both OD600nm to 0.1, and culturing for 24h in a constant temperature shaking table at 30 +/-2 ℃ and 150rpm to obtain a bacteria-containing water sample.

After 24h, in order to ensure that the fluorescence intensity is in a proper range, adding 2mL of phosphate buffer solution (0.1mM) into a culture pore plate of a 24-pore laser confocal scanning microscope for pre-balancing for 30min, then adding 500 μ L of a bacteria-containing water sample into the pore plate added with 2mL of phosphate buffer solution, and collecting a fluorescence image by using the laser confocal scanning microscope, wherein the parameter setting is consistent with the fluorescence intensity F-OD600nm absorbance standard curve image collection, the fluorescence image is shown in figure 3, A is pure water, B is a primary sedimentation tank, C is an adjusting tank, D is primary A/O effluent, and E is secondary A & lter & gtO is discharged, F is the inlet water of the disinfection tank, and G is the outlet water of the disinfection tank. Obtaining the fluorescence intensity in the water sample containing the bacteria, setting the fluorescence intensity as x, the OD600nm absorbance as y, and the bacteria concentration C as z (10)⁷cfu/mL), and obtaining the fluorescent Escherichia coli EGFP-BL21 receptor concentration C0 according to the previously obtained standard curve y of 0.0117x and z of 99.08 y-16.992.

Taking overnight-cultured green fluorescent escherichia coli EGFP-BL21 and drug-resistant Shigella (amoxicillin-resistant), adjusting the concentrations of the green fluorescent escherichia coli EGFP-BL21 and the drug-resistant Shigella to OD600nm ═ 0.6 by using nutrient broth, adding 10mL of bacterial liquid into 40mL of each to-be-detected membrane water sample, namely adding two bacteria of which the concentrations are OD600nm ═ 0.1 in the to-be-detected membrane water sample, and culturing for 24h in a constant temperature shaking table at 30 +/-2 ℃ and 150rpm to obtain a bacteria-containing water sample. Adding 1mL of a bacteria-containing water sample into 50mL of nutrient broth containing kanamycin (32 mu g/mL), amoxicillin (8 mu g/mL) and IPTG (0.1mmol/mL) (namely the content of the bacteria-containing water sample in the nutrient broth is 2 percent), and culturing for 24h in a constant temperature shaking table at the temperature of 30 +/-2 ℃ and the rpm of 150 to obtain a zygospore bacteria screening solution.

After 24h, in order to ensure that the fluorescence intensity is in a proper range, adding 2mL of phosphate buffer (0.1mM) into a 24-hole laser confocal scanning microscope culture hole plate for pre-balancing for 30min, then adding 2mL of zygospore bacteria screening solution into the hole plate, and similarly collecting fluorescence images by using a laser confocal scanning microscope, as shown in figure 4, A is pure water, B is a primary sedimentation tank, C is an adjusting tank, D is primary A/O effluent, E is secondary A/O effluent, F is disinfection tank inlet water, and G is disinfection tank outlet water. Measuring fluorescence intensity in the zygospore bacteria screening solution, wherein the fluorescence intensity is x, OD600nm absorbance is y, and the bacteria concentration C is z (10)⁷cfu/mL), and obtaining the number C of the zygotes according to the standard curve obtained in the previous period, wherein y is 0.0117x, and z is 99.08 y-16.992.

The migration frequency of the water sample resistance gene, namely the number of connexons, C/fluorescent escherichia coli EGFP-BL21 receptor C0, was plotted in fig. 5. As can be seen from FIG. 5, the resistance gene migration frequency of the effluent of the adjusting tank is the highest, and the resistance gene migration frequency of the effluent of the disinfection tank is the lowest, which is expected. 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 determine the migration frequency of the resistance genes under the water quality conditions (heavy metals, antibiotics and other organic substances) of various water samples to be detected.

The application selects fluorescent escherichia coli as a receptor, the fluorescent expression is stable, kanamycin resistance is contained, the screening is convenient, the laser confocal microscope is high in resolution, and the fluorescent escherichia coli and the laser confocal microscope can be combined and applied to quickly determine the migration frequency of the resistance genes in water quality to be detected after a standard curve is established, namely the comprehensive risk of the migration of the resistance genes in the water quality of a water sample to be detected.

The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.