CN117737185A - High-throughput screening method for antibacterial activity - Google Patents
High-throughput screening method for antibacterial activity Download PDFInfo
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- 238000013537 high throughput screening Methods 0.000 title claims abstract description 41
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a high-throughput screening method for antibacterial activity, and belongs to the technical field of antibacterial activity screening. Wherein the high throughput screening method of bacteriostatic activity comprises: taking a culture plate, and setting a negative control group, a positive control group and a drug experimental group aiming at a target strain in the culture plate to obtain a sample carrying plate; culturing the strain on the template to enable the target strain to reach a preset concentration; and (3) performing antibacterial activity detection on the cultured sample carrying plate. According to the invention, culture plates with different hole numbers are used as detection containers, the inhibition effect of one compound or medicine on various microorganisms is detected on one plate, and the Minimum Inhibitory Concentration (MIC) of various compounds or medicines can be detected simultaneously, so that high-throughput detection is realized. In addition, the method does not need to transfer samples, adopts instruments (such as ultraviolet rays, a microscope and the like) to directly measure the detection container, reduces pollution and reduces detection cost.
Description
Technical Field
The invention belongs to the technical field of antibacterial activity screening, and particularly relates to a high-throughput screening method for antibacterial activity.
Background
The antibacterial activity can evaluate the inhibition effect of a compound or a drug on microorganisms, thereby providing an important reference for new drug development and antibacterial agent screening. The antibacterial activity screening is the antibacterial activity evaluation process aiming at a novel compound, medicine or material. In bacteriostatic activity screening, the inhibitory effect of a target substance on a particular bacterium or fungus is typically assessed by a series of experiments or tests to determine if it has the potential for antimicrobial or bacteriostatic use. At present, most of antibacterial activity screening methods are long in time consumption and high in screening cost, so that a high-throughput screening method for antibacterial activity detection is urgently needed.
At present, the screening method of antibacterial activity can relate to a plurality of methods, mainly including oxford cup method, paper sheet diffusion method, conical flask method and microfluidic chip auxiliary method, and the screening methods are helpful for discovering new antibacterial agents or antibacterial materials and provide important reference information for the fields of drug research and development, medical instrument surface treatment, food preservation and the like. However, the above-described method still has a certain limitation.
The oxford cup method is a common antibacterial activity screening method, but the antibacterial effects of different oxford cups are greatly different, so that the accuracy of the result is affected; and the oxford cup method needs manual operation, is time-consuming and labor-consuming, is not suitable for high-throughput screening, and limits the application of the oxford cup method.
The paper sheet diffusion method is used for evaluating the antibacterial activity of the compound on the surface of the solid culture medium, and cannot evaluate the antibacterial activity of the compound in the liquid culture medium; the size of the antibacterial activity is estimated by the size of the antibacterial ring, the sensitivity is relatively low, and the method can only estimate the antibacterial activity of antibacterial substances in a short time; the method also needs manual operation, is time-consuming and labor-consuming, and is not suitable for high-throughput screening.
The micro-fluidic chip assisted antibacterial activity screening is a newly emerging screening method, and special micro-fluidic chips and equipment are needed, so that the chip size is small, the number and the volume of samples are limited, and the requirement of large-scale sample testing cannot be met; the special chip is required to be designed for specific microorganisms, and the cost is high.
In short, most of the existing antibacterial activity screening methods have the problems of long time consumption, inapplicability to high-throughput screening, time and labor consumption in manual operation, poor sensitivity, high cost and the like, so that a high-throughput screening method for detecting antibacterial activity is urgently needed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a high throughput screening method for antibacterial activity, comprising:
taking a culture plate, and setting a negative control group, a positive control group and a drug experimental group aiming at a target strain in the culture plate to obtain a sample carrying plate;
culturing the strain on the template to enable the target strain to reach a preset concentration;
and (3) performing antibacterial activity detection on the cultured sample carrying plate.
Preferably, the culture plate is selected from any one of a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, and a 6-well plate.
Preferably, each of the carrier plates includes a negative control group and a positive control group for the target strain, and at least one drug experimental group.
Preferably, the following areas are provided in the carrier plate for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 1-6 parallel holes are set for each concentration;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 1-6 hole sites in the same row;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, wherein 1-6 parallel holes are arranged in a same row;
preferably, the kit further comprises a fourth region which is a drug experimental group of the second drug to be tested on the first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 1-6 parallel holes are set for each concentration;
preferably, the first area, the second area, the third area and the fourth area are used as a test combination area;
when the culture plate is a 96-well plate, the culture plate comprises two test combination areas: one is a test combination region for the first strain of interest and the second is a test combination region for the second strain of interest.
Preferably, the following areas are provided in the carrier plate for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein, set up 3 concentrations, each said concentration sets up 4 parallel holes;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 4 hole sites;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, and is provided with 4 parallel holes;
preferably, the kit further comprises a fourth region which is a drug experimental group of the second drug to be tested on the first target strain; wherein, set up 3 concentrations, each said concentration sets up 4 parallel holes;
preferably, the first area, the second area, the third area and the fourth area are used as a test combination area;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
Preferably, the following areas are provided in the carrier plate for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a second area, which is a positive control group of the first target strain and consists of the first target strain and positive medicines, and is provided with 4 positive control groups; each hole site represents a concentration;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, and is provided with 4 parallel holes;
preferably, the method further comprises:
a fourth region, which is a drug experimental group of the second drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a fifth region, which is a drug experimental group of a third drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a sixth region, which is a drug experimental group of a fourth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a seventh region, which is a drug experimental group of a fifth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
an eighth region, which is a drug experimental group of a sixth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
preferably, the first region, the second region, the third region, the fourth region, the fifth region, the sixth region, the seventh region and the eighth region are one test combination region;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
Preferably, the total volume added per well in the negative control group, the positive control group and the drug experimental group is the same.
Preferably, each well of the negative control group, the positive control group and the drug test group contains at least 50. Mu.L of the target strain.
Preferably, the preset concentration is 0.001×10 5 CFU/mL-10×10 5 CFU/mL。
Preferably, in the step of detecting the antibacterial activity of the cultured sample carrying plate, the antibacterial activity detection includes obtaining an OD value corresponding to each hole in the sample carrying plate and/or observing the microbial condition.
The invention provides a high-throughput screening method for antibacterial activity. Wherein the method comprises the following steps: taking a culture plate, and setting a negative control group, a positive control group and a drug experimental group aiming at a target strain in the culture plate to obtain a sample carrying plate; culturing the strain on the template to enable the target strain to reach a preset concentration; and (3) performing antibacterial activity detection on the cultured sample carrying plate. The application provides a high-throughput screening method for antibacterial activity detection, which has the following beneficial effects:
(1) The detection container adopted by the method is small and convenient, can detect the antibacterial activity of different medicines on different microorganisms, and realizes high-flux detection;
(2) The application method has wide application range, can be used for most gram-negative bacteria and gram-positive bacteria, and cultures according to the growth characteristics of microorganisms;
(3) The culture medium is adopted to dilute the medicine, and then the medicine is added into the microorganism for culture, so that the sample is not required to be transferred for multiple times, secondary pollution is reduced, and the operation is convenient;
(4) The initial concentration of the test microorganism is adjusted according to the growth characteristics of the microorganism to ensure that the Minimum Inhibitory Concentration (MIC) is screened in a short time.
In a word, the invention uses the pore plates with different pore numbers as the detection container, and simultaneously detects the inhibition effect of one compound or medicine on various microorganisms on one plate, and simultaneously detects the Minimum Inhibitory Concentration (MIC) of various compounds or medicines, thereby realizing high-throughput detection. In addition, the method does not need to transfer samples, adopts instruments (such as ultraviolet rays, a microscope and the like) to directly measure the detection container, reduces pollution and reduces detection cost.
Drawings
FIG. 1 is a schematic flow chart of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 2 is a schematic layout of embodiment A of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 3 is a schematic diagram of a layout of a fourth region included in embodiment A of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 4 is a schematic layout diagram of two test combination areas in a 96-well plate according to embodiment A of the high throughput screening method of bacteriostatic activity of the present invention;
FIG. 5 is a schematic layout of embodiment B of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 6 is a schematic diagram of a layout of a fourth region included in embodiment B of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 7 is a schematic layout of three test combination zones in a 96-well plate according to embodiment B of the high throughput screening method of bacteriostatic activity of the present invention;
FIG. 8 is a schematic layout of embodiment C of a high throughput screening method of bacteriostatic activity according to the invention;
FIG. 9 is a schematic diagram showing a layout of a fourth region-eighth region included in embodiment C of a high throughput screening method of bacteriostatic activity according to the present invention;
FIG. 10 is a schematic layout of three test combination zones disposed in a 96-well plate according to embodiment C of the high throughput screening method of bacteriostatic activity of the present invention;
FIG. 11 is a schematic layout of example 1 of the high throughput screening method of bacteriostatic activity according to the invention;
FIG. 12 is a schematic layout of example 2 of the high throughput screening method of bacteriostatic activity according to the invention.
Reference numerals:
1, carrying a template; 11, a first test combination zone; 12, a second test combination zone; 13, a third test combination zone; 111, a first region; 112, a second region; 113, a third region; 114, a fourth region; 115, a fifth region; 116, a sixth region; 117, seventh region; 118, eighth region; 1111, holes.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1, the present invention provides a high throughput screening method of bacteriostatic activity, comprising:
step S1, taking a culture plate, and setting a negative control group, a positive control group and a drug experimental group aiming at a target strain in the culture plate to obtain a sample carrying plate;
the culture plate is a biochemical experimental method, and is mainly used for measuring the content of specific proteins, antigens, antibodies or hormones in a liquid sample. The method is very sensitive and has strong specificity, and is widely applied to the fields of medicine, biology, bioengineering and the like.
The plates may have different numbers of wells, and may be, for example, 96-well plates.
In the screening test of antibacterial activity, negative control, positive control and drug group are set to ensure accuracy and reliability of test results. Each control group had its specific effect:
(1) Negative control: generally comprising a microbial culture without any antimicrobial added. The purpose of this control group was to demonstrate the growth of microorganisms without any antibacterial intervention. From this control, it was confirmed that the experimental conditions themselves (e.g., medium, temperature, etc.) had no adverse effect on the growth of microorganisms. At the same time, it also provides a baseline data for comparison with the effects of other experimental groups (e.g., drug groups).
(2) Positive control: including known substances having bacteriostatic activity, such as certain antibiotics, which have proven effective. The purpose of the positive control is to demonstrate that the experimental conditions are suitable for detection of bacteriostatic activity. If the positive control group shows the expected antibacterial effect, the experimental design is correct, and the activity of the antibacterial substance can be effectively detected. Meanwhile, the positive control also provides a reference for evaluating the antibacterial strength of the drug to be tested.
(3) Drug group: this is the main part of the experiment and includes the antimicrobial substance to be tested. The bacteriostatic activity potential of the test substance can be assessed by comparing the results of the drug group with negative and positive controls. If the drug group shows similar antibacterial effect as the positive control, the substance to be tested has antibacterial activity. If the effect is close to the negative control, the substance to be detected has no obvious antibacterial effect.
In summary, by setting the three control groups, the antibacterial activity of the substance to be tested can be more accurately estimated, and meanwhile, the reliability and the accuracy of the experimental result are ensured.
The culture plate is used for detection in the antibacterial activity experiment, and the detection result is obtained through the enzyme-labeled instrument, so that the method has a plurality of advantages:
the sensitivity is high: the microplate reader is capable of detecting color changes in the culture plate very sensitively, and even very weak signals can be read accurately. This allows even low concentrations of bacteriostats or weak changes in microbial growth to be detected.
And (3) quantitative accuracy: the microplate reader provides a quantitative reading, which means that the intensity of bacteriostatic activity in different samples can be accurately measured and compared. This is important for applications such as determining the Minimum Inhibitory Concentration (MIC) and plotting the dose-response curve of the effect of the bacteriostatic agent.
Quick and convenient: the detection result of a large number of samples can be obtained rapidly by using the enzyme-labeled instrument, and the experimental efficiency is improved greatly. This is particularly useful for screening studies where a large number of samples need to be processed.
Automation and standardization: the microplate reader can be generally used in combination with computer software to enable automated collection and analysis of data. This helps to reduce human error, ensuring consistency and repeatability of the data.
The applicability is wide: besides being used for antibacterial activity test, the culture plate and the enzyme-labeled instrument are widely applied to various fields of biochemistry, molecular biology, immunology and the like, and the flexibility and the versatility of the culture plate and the enzyme-labeled instrument are shown.
Minimizing sample usage: the culture plate allows experiments to be performed in a very small volume, which can reduce the amount of reagents and samples required and reduce the cost of the experiments.
In a word, the antibacterial activity test is carried out by using the culture plate and the enzyme-labeled instrument, so that the sensitivity and the accuracy of detection are improved, the efficiency and the standardization degree of the experiment are also improved, and the method is a very effective experiment method.
Step S2, culturing the strain on the sample carrying plate to enable the target strain to reach a preset concentration;
and S3, detecting the antibacterial activity of the cultured sample carrying plate.
It should be noted that, in the antibacterial activity screening test, different experimental groups are set on a culture plate to perform culture, and the strain is detected after reaching a preset concentration, which has several key reasons and advantages:
ensure the activity of the strain: by culturing, it was possible to ensure that the strain used in the experiment was active and had a representative growth state. This is critical for assessing the effect of the bacteriostatic agent, as the response of a dead or dormant strain to a drug may not be representative.
Establishing consistent starting conditions: before starting the antibacterial activity test, all strains are allowed to reach the same preset concentration, so that comparability among different experimental groups can be ensured. This is because the growth phase of the microorganisms (e.g., lag phase, log phase, or stationary phase) affects their sensitivity to the antimicrobial agent.
Enhancing the accuracy and repeatability of the experiment: by controlling the initial concentration of the strain, experimental variation can be reduced, and the accuracy and repeatability of the result can be improved. This allows for a more reliable comparison of differences between different experimental groups.
Convenient dose effect analysis: when the strain reaches a preset concentration, the influence of the bacteriostat at different concentrations on the growth of microorganisms can be analyzed more accurately, so that the Minimum Inhibitory Concentration (MIC) and other related parameters can be determined accurately.
Evaluating the dynamic effect of the bacteriostat: by monitoring the growth conditions of the bacterial strain at different time points, the dynamic inhibition effect of the bacteriostat can be known, such as whether the delayed inhibition effect exists or whether the action speed of the bacteriostat exists or not.
In summary, in the antibacterial activity screening test, the strain is cultured to a certain concentration on a culture plate and then detected, and the step is to ensure the accuracy, reliability and comparability of the test.
The detection method can adopt ultraviolet or enzyme-labeled instrument to detect the OD value of microorganism growth, and adopts microscope to observe the growth condition of microorganism.
The embodiment provides a high-throughput screening method for detecting antibacterial activity, which uses culture plates with different hole numbers as detection containers, simultaneously detects the inhibition effect of one compound or medicine on various microorganisms on one plate, and simultaneously detects the Minimum Inhibitory Concentration (MIC) of various compounds or medicines, thereby realizing high-throughput detection. In addition, the method does not need to transfer samples, adopts instruments (such as ultraviolet rays, a microscope and the like) to directly measure the detection container, reduces pollution and reduces detection cost.
Further, the culture plate can be any one of the following specifications based on the number of holes:
96-well plates, 48-well plates, 24-well plates, 12-well plates, or 6-well plates.
The number of wells of the culture plate may be selected according to the experimental requirements.
Further, each carrier plate comprises a negative control group and a positive control group for the target strain, and at least one drug experimental group based on the drug to be tested of the target strain.
In the sample loading plate, a negative control group, a positive control group and a drug experimental group are required to be respectively set, wherein a negative control group and a positive control group of one group are required to be tested for the drug experimental group. The drug test groups can be set with different drug groups according to the experimental screening requirement, for example, in one sample carrying plate, there are 1 negative control group, 1 positive control group and 5 drug test groups of 5 drugs B, D, E, F, G to be tested for the strain A.
For the culture plate, the following embodiments are set in the invention:
1. embodiment a:
referring to fig. 2, in this embodiment, at least one of the following regions for the target strain is provided in the carrier plate:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 6 parallel wells are set for each concentration;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 6 hole sites in a same row;
and the third area is a negative control group of the first target strain, consists of the first target strain and negative medicines, and is provided with 6 parallel holes in a same row.
In one of the culture plates, the following may be set up:
first, 3 regions were set, which are the first region, the second region, and the third region for 1 target strain, respectively. The 3 areas correspond to the drug experimental group, the positive control group and the negative control group, respectively.
The above configuration is that the most basic configuration for performing antibacterial activity screening experiments for one target strain is set in one culture plate, and a single drug is specifically tested, so that a group of drug experiment groups is adopted, and if multiple drugs are tested, the setting of the multiple drug experiment groups can be performed according to the above setting requirements.
The first zone may be set to 3 different concentrations, one concentration per row, 6 parallel wells per concentration, or 1 row, with each well representing one concentration.
The second region and the third region may each be arranged in a row of 6 hole sites each.
Further, referring to fig. 3, in this embodiment, a fourth region may be added in addition to the first region, the second region, and the third region, which is a drug experimental group of the second drug to be tested for the first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 6 parallel wells are set for each concentration;
in the above-mentioned multi-drug activity test, the above-mentioned mode may be adopted, that is, one more drug test group is set, and the same is 3 concentration gradients, and 6 hole sites are set for each concentration gradient.
Further, referring to fig. 4, the first area, the second area, the third area and the fourth area are taken as a test combination area;
when the culture plate is a 96-well plate, the culture plate comprises two test combination areas: one is a test combination region for the first strain of interest and the second is a test combination region for the second strain of interest.
In the 96-well plate, the first region, the second region, the third region and the fourth region together form a test combination region. The specific settings may be as follows:
in a plate, if a single test combination zone is used, a test combination zone 1 may be provided: the second area and the third area are respectively taken as a positive control group and a negative control group of the target strain A, the first area is taken as a drug experimental group of a first drug to be tested, and the fourth area is taken as a drug experimental group of a second drug to be tested.
In a culture plate, such as a 96-well plate, two test combination areas can be simultaneously arranged, and besides the test combination area 1, the following arrangement can be performed:
another test combination zone 2 may be: the sixth area and the seventh area are respectively taken as a positive control group and a negative control group of the target strain D, the fifth area is taken as a drug experimental group of a second drug to be tested, and the eighth area is taken as a drug experimental group of a fourth drug to be tested.
The positions of the areas can be changed, and only one arrangement mode is provided in the embodiment.
2. Embodiment B:
referring to fig. 5, in this embodiment, at least one of the following regions for the target strain is provided in the carrier plate:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein 3 concentrations are set, and 4 parallel wells are arranged for each concentration gradient;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 4 hole sites;
and the third area is a negative control group of the first target strain, consists of the first target strain and a negative medicament, and is provided with 4 parallel holes.
In this example, the above embodiment a is modified.
Wherein the second region and the third region are also a positive control group and a negative control group for the target strain, and each of the 6 sites is reduced to 4 sites.
The first region, which is a drug experimental group of the first drug to be tested for the first target strain, may be set to 3 concentrations, with 4 parallel wells for each concentration gradient as well.
Further, referring to fig. 6, a fourth region is also included, which is a drug experimental group of the second drug to be tested against the first target strain; wherein, set up 3 concentrations, each said concentration sets up 4 parallel holes;
further, referring to fig. 7, the first area, the second area, the third area and the fourth area are taken as a test combination area;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
3. Embodiment C:
referring to fig. 8, at least one of the following regions for the target strain is provided in the culture plate:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 4 hole sites; each hole site represents a concentration;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, and is provided with 4 parallel holes;
further, referring to fig. 9, further includes:
a fourth region, which is a drug experimental group of the second drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a fifth region, which is a drug experimental group of a third drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a sixth region, which is a drug experimental group of a fourth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a seventh region, which is a drug experimental group of a fifth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
an eighth region, which is a drug experimental group of a sixth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
further, referring to fig. 10, the first area, the second area, the third area, the fourth area, the fifth area, the sixth area, the seventh area, and the eighth area are taken as one test combination area;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
Further, the total volume added per well in the negative control group, the positive control group and the drug experimental group was the same.
In the antibacterial activity screening experiment, the same total volume added in each hole in the negative control group, the positive control group and the drug experiment group is ensured to ensure comparability of the experiment and accuracy of the result.
By ensuring that the total volume added in each hole in the negative control group, the positive control group and the drug experimental group is the same, experimental errors can be reduced as much as possible, and the reliability and the repeatability of experimental results can be improved. Therefore, the antibacterial activity of the medicine can be more effectively evaluated, and reasonable comparison and analysis can be performed.
Further, each well of the negative control group, the positive control group and the drug test group contains at least 50. Mu.L of the target strain.
Further, the preset concentration is 0.001×10 5 CFU/mL-10×10 5 CFU/mL。
Further, in the step of detecting the antibacterial activity of the cultured sample carrying plate, the antibacterial activity detection includes obtaining an OD value corresponding to each hole in the sample carrying plate and/or observing the microbial condition.
As mentioned above, OD is an abbreviation for Optical Density (Optical Density) and is used to measure the absorption of a substance in a solution for light of a particular wavelength. In microbiology and cell culture, OD values are often used to evaluate bacterial, fungal or cell growth. In bacteriostatic activity screening experiments, obtaining OD values of the plates usually requires the use of microplate readers (microplate reader) or enzyme-labeled instruments (ELISA reader). These devices can measure the optical density (OD value) of microbial growth in the culture plates to assess bacteriostatic activity.
As mentioned above, in bacteriostatic activity screening experiments, the following equipment is generally required to observe the status of microorganisms on a culture plate:
and (3) a microscope: a microscope is an instrument for magnifying and observing minute objects. By placing under the microscope lens under the culture plate, the shape, structure and quantity characteristics of bacteria, fungi or other microorganisms can be observed.
Incubator: an incubator is a device that controls temperature and humidity to provide suitable environmental conditions to support the growth of microorganisms. In performing the observation of microorganisms on the culture plate, the incubator is set to an appropriate temperature and humidity condition to maintain the living state of the microorganisms.
Culture dish carousel: the dish carousel is a rotatable device for facilitating the observation of the growth of microorganisms on a plurality of dishes. By placing the culture dish on the turntable, the dish can be easily turned and positioned for observing microorganisms at different locations.
An image recording apparatus: in order to record and save the results of the microorganism observation, a camera, a digital video camera, or image capturing software, etc. may be used. These devices can help record information about the morphology, distribution and quantity of microorganisms on the culture plate.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1:
in this embodiment, according to the high-throughput screening method of bacteriostatic activity in the present invention, the following settings are performed:
96-well culture plates were selected for drug A and drug B, respectively.
Specifically, referring to fig. 11, the following settings are made:
(1) The first area and the fifth area are set as a tested sample group of the drug A to be tested for the escherichia coli, each row represents one concentration, and the total concentration of the drug A to be tested is 3.
(2) The second region and the sixth region are positive control groups of escherichia coli, and consist of escherichia coli and positive medicines.
(3) The third and seventh regions are a negative control group of E.coli, consisting of E.coli.
(4) The fourth and eighth regions are sample groups of drug B to be tested against E.coli, each row representing one concentration, drug B totaling 3 concentrations.
The parameters of the specific areas are as follows:
TABLE 1 drug screening model parameters for inhibiting E.coli
The composition of each region was added according to the above table and incubated overnight at 37 ℃.
The absorbance was measured at 600nm on a microplate reader and the bacteriostasis was then calculated according to the following formula:
antibacterial ratio (%) = (a negative control group-a test sample group)/a negative control group × 100%.
Experimental results:
(1) In the first area and the fourth area, the inhibition rates of the drug A to be tested and the drug B to be tested on the escherichia coli are 1% and 2%, respectively, and the inhibition rate of ceftriaxone sodium in the area 2 on the escherichia coli is 95%.
(2) In the fifth region and the eighth region, the inhibition rates of the drug A to be detected and the drug B to be detected on the escherichia coli are respectively 60% and 75%, and the inhibition rates and the drug concentration form positive correlation; the inhibition rate of ceftriaxone sodium to the escherichia coli in the region six is 96%.
The experimental results show that the medicine A to be tested and the medicine B to be tested have a certain inhibition effect on escherichia coli, and the concentration of the strain in the first area is too high, so that the medicine cannot fully exert the antibacterial activity. Therefore, the concentration of the tested strain needs to be fully considered in the bacteriostasis experiment.
Example 2:
in this embodiment, according to the high-throughput screening method of bacteriostatic activity in the present invention, the following settings are performed:
a 24-well plate was selected for drug C to be tested.
Specifically, referring to fig. 12, the following settings are made:
(1) The first region is a sample group of the drug C to be tested against bacillus subtilis, each column represents one concentration, and the total concentration of the drug C to be tested is 4.
(2) The second region is a positive control group of bacillus subtilis, which consists of bacillus subtilis and positive drugs.
(3) The third region is a negative control group of bacillus subtilis, consisting of bacillus subtilis. The specific parameters of each region are as follows:
TABLE 2 drug screening model parameters for inhibition of Bacillus subtilis
The composition of each region was added according to the above table and incubated overnight at 37 ℃.
Observing the growth condition of bacillus subtilis under a microscope, judging to be bacteriostatic according to the conditions of damaged cell walls, thinned cell walls or locally obvious damage, deformed cells and the like, and then calculating the bacteriostatic rate according to the following formula:
antibacterial ratio (%) =the number of damaged bacillus subtilis in the test sample group/the number of bacillus subtilis in the negative control group ×100%.
Experimental results:
the results show that the experiment only needs to determine the proper concentration of the tested strain, the growth state of the tested strain is observed by means of an optical microscope, and the statistics of the experimental result can be completed without other transfer operations, so that the risk of pollution is reduced to the maximum extent, the detection time is saved, and the detection efficiency is improved.
The embodiment provides a high-throughput screening method for detecting antibacterial activity, which uses pore plates with different pore numbers as detection containers, simultaneously detects the inhibition effect of one compound or medicine on various microorganisms on one plate, and simultaneously detects the Minimum Inhibitory Concentration (MIC) of various compounds or medicines, thereby realizing high-throughput detection. In addition, the method does not need to transfer samples, adopts instruments (such as ultraviolet rays, a microscope and the like) to directly measure the detection container, reduces pollution and reduces detection cost.
Compared with the traditional antibacterial activity screening, the method can finish screening of various microorganisms and various medicines in one detection container, so that high-throughput screening is realized; the antibacterial detection time is shortened by optimizing the initial strain concentration; meanwhile, by combining with ultraviolet detection and other instruments, the error of the experimental result is greatly reduced; and the sample is not required to be transferred again or the sample is repeatedly sampled during detection, so that secondary pollution of the sample is avoided.
While the preferred embodiments and examples of the present invention have been described, it should be noted that modifications and improvements, including but not limited to, adjustments of proportions, procedures, amounts and reaction vessels, may be made by those skilled in the art without departing from the inventive concept herein.
Claims (10)
1. A high throughput screening method for bacteriostatic activity, comprising:
taking a culture plate, and setting a negative control group, a positive control group and a drug experimental group aiming at a target strain in the culture plate to obtain a sample carrying plate;
culturing the strain on the template to enable the target strain to reach a preset concentration;
and (3) performing antibacterial activity detection on the cultured sample carrying plate.
2. The high throughput screening method of bacteriostatic activity according to claim 1, wherein said culture plate is selected from any one of 96-well plate, 48-well plate, 24-well plate, 12-well plate and 6-well plate.
3. The high throughput screening method of bacteriostatic activity according to claim 1, wherein each of said carrier plates comprises a negative control group and a positive control group for said target strain and at least one drug experimental group.
4. A high throughput screening method for bacteriostatic activity according to claim 3, characterized in that in said carrier plate there are provided the following regions for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 1-6 parallel holes are set for each concentration;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 1-6 hole sites in the same row;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, wherein 1-6 parallel holes are arranged in a same row;
preferably, the kit further comprises a fourth region which is a drug experimental group of the second drug to be tested on the first target strain; wherein each row represents one concentration, 3 concentrations are set in total, and 1-6 parallel holes are set for each concentration;
preferably, the first area, the second area, the third area and the fourth area are used as a test combination area;
when the culture plate is a 96-well plate, the culture plate comprises two test combination areas: one is a test combination region for the first strain of interest and the second is a test combination region for the second strain of interest.
5. The high throughput screening method of bacteriostatic activity according to claim 4, wherein the following regions are provided in said carrier plate for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein, set up 3 concentrations, each said concentration sets up 4 parallel holes;
the second area is a positive control group of the first target strain, consists of the first target strain and a positive medicament, and is provided with 4 hole sites;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, and is provided with 4 parallel holes;
preferably, the kit further comprises a fourth region which is a drug experimental group of the second drug to be tested on the first target strain; wherein, set up 3 concentrations, each said concentration sets up 4 parallel holes;
preferably, the first area, the second area, the third area and the fourth area are used as a test combination area;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
6. The high throughput screening method of bacteriostatic activity according to claim 4, wherein the following regions are provided in said carrier plate for at least one target strain:
the first area is a drug experimental group of a first drug to be tested on a first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a second area, which is a positive control group of the first target strain and consists of the first target strain and positive medicines, and is provided with 4 positive control groups; each hole site represents a concentration;
a third area, which is a negative control group of the first target strain and consists of the first target strain and a negative medicament, and is provided with 4 parallel holes;
preferably, the method further comprises:
a fourth region, which is a drug experimental group of the second drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a fifth region, which is a drug experimental group of a third drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a sixth region, which is a drug experimental group of a fourth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
a seventh region, which is a drug experimental group of a fifth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
an eighth region, which is a drug experimental group of a sixth drug to be tested on the first target strain; wherein each hole site represents one concentration, and 4 concentrations are set in total;
preferably, the first region, the second region, the third region, the fourth region, the fifth region, the sixth region, the seventh region and the eighth region are one test combination region;
when the culture plate is a 96-well plate, 3 of the test combination zones are included in the culture plate: one is a test combination region for the first target strain, the second is a test combination region for the second target strain, and the third is a test combination region for the third target strain.
7. The high throughput screening method of bacteriostatic activity according to claim 1, wherein the total volume added per well in said negative control group, said positive control group and said drug experimental group is the same.
8. The high throughput screening method of bacteriostatic activity according to claim 1, wherein each well of said negative control group, said positive control group and said drug test group contains at least 50 μl of said target strain.
9. The high throughput screening method of antibacterial activity according to claim 1, wherein said predetermined concentration is 0.001×10 5 CFU/mL-10×10 5 CFU/mL。
10. The method for high-throughput screening of antibacterial activity according to claim 1, wherein in the step of detecting antibacterial activity of the cultured carrier plate, antibacterial activity detection comprises obtaining an OD value corresponding to each hole in the carrier plate and/or observing microbial conditions.
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