Method for screening bacterial strains with antibacterial function

Abstract

The invention discloses a method for screening bacterial strains with an antibacterial function, which comprises the following steps: to-be-screened bacteria and indicator bacteria are screened in a microfluidic system, the indicator bacteria have a fluorescent label so as to obtain target bacteria, and the target bacteria have the activity of inhibiting the indicator bacteria. According to the method, the antagonistic activity of the strains is associated with the fluorescence signal intensity, the functional strains with the antibacterial function are screened in a high-throughput and high-precision mode, the screening throughput reaches 106-108 strains every day, and therefore the screening efficiency and the result stability are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology. Specifically, the present invention relates to a method for screening strains with antibacterial function, and more specifically, the present invention relates to a method for high-throughput screening of strains with antibacterial function based on picoliter droplet system combined with fluorescence signal intensity. Background Art

[0002] Antibacterial functional strains refer to microbial strains that have the ability to inhibit the growth and reproduction of other microorganisms. These strains usually produce one or more substances with antibacterial activity, such as antibiotics, antibacterial peptides, enzymes, etc., which are used to compete with potential pathogenic microorganisms or directly kill them.

[0003] Traditional methods for screening strains with antibacterial function are to screen antibacterial strains by combining the filter paper method with the generation of antibacterial zones on the culture medium plate with indicator bacteria. The screening throughput is limited by manual operation, and the reaction system of this method is in the milliliter level. Traditional methods for screening strains with antibacterial function can also be achieved by using multi-well plates combined with colony area scanners, but the screening throughput is limited by the number of wells of the plate, and the reaction system of this method is in the microliter level.

[0004] Traditional methods for screening strains with antibacterial function have the disadvantages of low throughput, low precision, and high cost. Therefore, there is an urgent need to develop a method for screening antibacterial functional strains with high throughput, high precision, and low cost. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent.

[0006] The inventors found that the prior art cannot effectively screen out functional strains with antibacterial function from a complex sample library. To overcome this problem, the inventors used a microfluidic system to develop a method for screening functional strains with antibacterial function by correlating fluorescence signal intensity with antibacterial activity, and efficiently screened out a class of functional strains that inhibit a certain bacterium from a complex sample library. The screening throughput of this method for screening functional strains with antibacterial function can reach 10 6 ~10 8 strains per day.

[0007] Based on this, in one aspect of the present invention, the present invention provides a method for screening strains with antibacterial function. According to an embodiment of the present invention, the method includes subjecting the bacteria to be screened and the indicator bacteria to screening treatment in a microfluidic system, wherein the indicator bacteria have a fluorescent label, so as to obtain target bacteria, and the target bacteria have the activity of inhibiting the indicator bacteria. This method correlates the antagonistic activity of the strains with the fluorescence signal intensity, and realizes high-throughput and high-precision screening of functional strains with antibacterial function, and the screening throughput reaches 10 6 ~108 strains, thus significantly improving the screening efficiency and the stability of the results.

[0008] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0010] Figure 1 It is a step diagram for screening strains with antibacterial functions using a microfluidic system in Example 1.

[0011] Figure 2 It is a schematic diagram of the generation of the first droplet in Example 1.

[0012] Figure 3 It is a bright-field microscopy image of the second droplet in Example 1.

[0013] Figure 4 It is a schematic diagram of microinjecting the indicator bacteria into the second droplet in Example 1.

[0014] Figure 5 It is a schematic diagram of quality control of the third droplet through a fluorescence threshold in Example 1.

[0015] Figure 6 It is a fluorescence field microscopy image of the constant-temperature culture of the third droplet in Example 1.

[0016] Figure 7 It is a microscopy image and a plate verification image after fluorescence sorting of the fourth droplet in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0017] Embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0018] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in the present invention, all other technical and scientific terms used in the present invention have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0019] In the present invention, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present invention, but does not exclude other aspects.

[0020] In the present invention, the terms "optionally", "optional" or "option" generally mean that the subsequent event or condition may but does not necessarily occur, and this description includes the case where the event or condition occurs and the case where the event or condition does not occur.

[0021] It should be noted that the terms "first" and "second" are only for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality of" means two or more.

[0022] In one aspect of the present invention, a method for screening strains with antibacterial function is proposed. According to an embodiment of the present invention, the method includes subjecting the bacteria to be screened and the indicator bacteria to screening treatment in a microfluidic system, wherein the indicator bacteria have a fluorescent label so as to obtain the target bacteria, and the target bacteria have the activity of inhibiting the indicator bacteria. This method correlates the antagonistic activity of the strains with the fluorescence signal intensity, realizing high-throughput and high-precision screening of functional strains with antibacterial function, and the screening throughput reaches 10 6 ~10 8 strains per day, thus significantly improving the screening efficiency and the stability of the results.

[0023] According to an embodiment of the present invention, the microfluidic system includes a droplet generation chip, a micro-injection chip and a droplet sorting chip.

[0024] According to an embodiment of the present invention, the screening process is carried out in the following manner: the bacteria to be screened are subjected to a first droplet generation process to obtain first droplets, and each of the first droplets contains one strain of the bacteria to be screened; the first droplets are subjected to a first culture process to obtain second droplets; the indicator bacteria are micro-injected into the second droplets to obtain third droplets; based on the first fluorescence signal of the third droplets, the third droplets are subjected to a second culture process to obtain fourth droplets; based on the second fluorescence signal of the fourth droplets, target fourth droplets are obtained, and the target bacteria are contained in the target fourth droplets. According to an embodiment of the present invention, each of the second droplets contains one of the bacteria to be screened amplified from one strain of the bacteria to be screened. By micro-injecting the indicator bacteria into the second droplets, it is ensured that each of the third droplets contains only one strain of the indicator bacteria. Based on the first fluorescence signal of the third droplets, it is determined whether the third droplets contain indicator bacteria. The third droplets containing indicator bacteria are continuously cultured. During the continuous culture process, an equivalent nutritional competition relationship between the indicator bacteria and the bacteria to be screened is achieved, with a suitable bacterial liquid concentration, combined with the amplification of the strains in the appropriate droplets, so that the amount of the bacteria to be screened in the droplets is equivalent to the amount of the indicator bacteria, excluding the possibility of the influence of nutritional competition on the antagonistic reaction activity. The screening process of the embodiment of the present invention maximally realizes that each of the third droplets and the fourth droplets contains one of the bacteria to be screened and the indicator bacteria. Through droplet micro-injection and fluorescence sorting, it is ensured that the antagonistic reaction between the bacteria to be screened and the indicator bacteria has a high proportion, achieving more precise control and operation, and improving the accuracy of screening for antibacterial strains.

[0025] According to an embodiment of the present invention, before the first droplet generation process, it further includes pre-culturing the bacteria to be screened. After the pre-culturing of the bacteria to be screened, the OD600 of the bacteria to be screened is 0.03 - 0.05, such as 0.03, 0.035, 0.04, 0.045, 0.05, and the range values between any two of these point values, such as 0.03 - 0.045, 0.03 - 0.04, 0.03 - 0.035. The inventors found that the appropriate OD600 of the bacteria to be screened is beneficial to the amplification of the strains of the bacteria to be screened in the droplets, thereby achieving an equivalent nutritional competition relationship between the bacteria to be screened and the indicator bacteria, and excluding the possibility of the influence of nutritional competition on the antagonistic reaction activity between the bacteria to be screened and the indicator bacteria.

[0026] According to an embodiment of the present invention, before injecting the indicator bacterium into the second droplet, it further includes performing pre-culture treatment on the indicator bacterium. After the pre-culture treatment of the indicator bacterium, the OD600 of the indicator bacterium is 0.4 to 0.8. For example, 0.4, 0.5, 0.6, 0.7, 0.8, and the range values between any two of these point values, such as 0.4 to 0.7, 0.5 to 0.7, 0.6 to 0.7. The inventors found that a suitable OD600 of the indicator bacterium is beneficial to achieving an equivalent nutritional competition relationship between the indicator bacterium and the bacterium to be screened, and excluding the possibility of the influence of nutritional competition on the antagonistic reaction activity between the bacterium to be screened and the indicator bacterium.

[0027] According to an embodiment of the present invention, before injecting the indicator bacterium into the second droplet, it further includes performing pre-culture treatment on the indicator bacterium. After the pre-culture treatment of the indicator bacterium, the OD600 of the indicator bacterium is 0.6. A more suitable OD600 of the indicator bacterium is more beneficial to achieving an equivalent nutritional competition relationship between the indicator bacterium and the bacterium to be screened, and excluding the possibility of the influence of nutritional competition on the antagonistic reaction activity between the bacterium to be screened and the indicator bacterium.

[0028] According to an embodiment of the present invention, the first droplet generation treatment is performed in the droplet generation chip.

[0029] According to an embodiment of the present invention, the diameter of the first droplet is 10 to 50 μm, and the generation rate of the first droplet is 10 6 ~10 8 pieces / h. Compared with the prior art strain screening system at the milliliter / microliter level, the screening system of the embodiment of the present invention is upgraded to the picoliter level, achieving more precise control and operation, and improving the accuracy and screening throughput of the antibacterial strain screening.

[0030] According to an embodiment of the present invention, the diameter of the first droplet is 10 to 50 μm, and the generation rate of the first droplet is 10 7 pieces / h. Compared with the prior art strain screening system at the milliliter / microliter level, the screening system of the embodiment of the present invention can be further upgraded to the picoliter level, achieving more precise control and operation, and further improving the accuracy and screening throughput of the antibacterial strain screening.

[0031] According to an embodiment of the present invention, the first culture treatment is performed at a temperature of 35 to 42 °C for 1 to 5 h. This first culture treatment enables the bacterium to be screened to be cultured and amplified in the first droplet, so as to achieve an equivalent nutritional competition relationship between the bacterium to be screened and the indicator bacterium, and exclude the possibility of the influence of nutritional competition on the antagonistic reaction activity between the bacterium to be screened and the indicator bacterium.

[0032] According to an embodiment of the present invention, the first culturing treatment is carried out at a temperature of 37 °C for 2 h. This first culturing treatment enables the bacteria to be screened to be cultured and amplified in the first droplet, so as to better achieve an equivalent nutritional competition relationship between the bacteria to be screened and the indicator bacteria, and eliminate the possibility of the influence of nutritional competition on the antagonistic reaction activity between the bacteria to be screened and the indicator bacteria.

[0033] According to an embodiment of the present invention, the first culturing treatment is carried out in the presence of at least one of fluorinated oil, mineral oil, and silicone oil.

[0034] According to some alternative embodiments of the present invention, the first culturing treatment is carried out in the presence of fluorinated oil.

[0035] According to an embodiment of the present invention, injecting the indicator bacteria into the second droplet is carried out in the micro-injection chip. To ensure that each second droplet contains only one type of indicator bacteria.

[0036] According to an embodiment of the present invention, the first fluorescence signal of the third droplet is obtained in the droplet sorting chip.

[0037] According to an embodiment of the present invention, the screening rate based on the first fluorescence signal of the third droplet is 10 4 ~10 6 per hour. In the embodiment of the present invention, the screening rate based on the first fluorescence signal of the third droplet is improved to the picoliter level compared with the milliliter / microliter strain screening system in the prior art, realizing more precise control and operation, and improving the accuracy and screening throughput of antibacterial strain screening.

[0038] According to an embodiment of the present invention, the screening rate based on the first fluorescence signal of the third droplet is 10 5 per hour. In the embodiment of the present invention, the screening rate based on the first fluorescence signal of the third droplet is improved to the picoliter level compared with the milliliter / microliter strain screening system in the prior art, further realizing more precise control and operation, and further improving the accuracy and screening throughput of antibacterial strain screening.

[0039] According to an embodiment of the present invention, the fluorescence sorting range of the first fluorescence signal is 3.5 to 10. According to an embodiment of the present invention, the third droplets with a fluorescence signal value of 3.5 to 10 of the first fluorescence signal can be regarded as successfully micro-injected indicator bacteria, and the third droplets with a first fluorescence signal of 3.5 to 10 are collected for subsequent second culture treatment. According to an embodiment of the present invention, by using fluorescence screening technology, the amount of indicator bacteria in the third droplets after micro-injection is quality-controlled to ensure the uniformity of the amount of indicator bacteria in the third droplets after micro-injection, so that the third droplets can undergo sufficient symbiotic or antagonistic reactions subsequently, and further ensure that the change in fluorescence intensity in the subsequent fourth droplets is mainly affected by the antagonistic reaction, thereby ensuring the correlation between low-value fluorescence and high antagonistic activity and the reliability of the results.

[0040] According to an embodiment of the present invention, the second culture treatment is carried out at a temperature of 35 to 42 °C for 15 to 20 h. The second culture treatment according to an embodiment of the present invention enables the indicator bacteria and the bacteria to be screened to undergo sufficient symbiosis or antagonism, and further ensures that the change in fluorescence intensity in the fourth droplets is mainly affected by the antagonistic reaction, thereby ensuring the correlation between low-value fluorescence and high antagonistic activity and the reliability of the results.

[0041] According to an embodiment of the present invention, the second culture treatment is carried out at a temperature of 37 °C for 18 h. The second culture treatment according to an embodiment of the present invention is more capable of enabling the indicator bacteria and the bacteria to be screened to undergo sufficient symbiosis or antagonism, and further ensures that the change in fluorescence intensity in the fourth droplets is mainly affected by the antagonistic reaction, thereby ensuring the correlation between low-value fluorescence and high antagonistic activity and the reliability of the results.

[0042] According to an embodiment of the present invention, the second culture treatment is carried out in the presence of at least one of fluorinated oil, mineral oil, and silicone oil.

[0043] According to some alternative embodiments of the present invention, the second culture treatment is carried out in the presence of fluorinated oil.

[0044] According to an embodiment of the present invention, the second fluorescence signal of the fourth droplets is obtained in a droplet sorting chip.

[0045] According to an embodiment of the present invention, the screening rate based on the second fluorescence signal of the fourth droplets is 10 4 ~10 6 per hour. In the embodiment of the present invention, the screening rate based on the second fluorescence signal of the fourth droplets is improved to the picoliter level compared with the milliliter / microliter strain screening system in the prior art, realizing more precise control and operation, and improving the accuracy and screening throughput of antibacterial strain screening.

[0046] According to an embodiment of the present invention, the screening rate based on the second fluorescence signal of the fourth droplet is 10 5 per hour. In the embodiment of the present invention, the screening rate based on the second fluorescence signal of the fourth droplet is increased to the picoliter level compared with the milliliter / microliter strain screening system in the prior art, realizing more precise control and operation, and improving the accuracy and screening throughput of the antibacterial strain screening.

[0047] According to an embodiment of the present invention, the fluorescence sorting threshold of the second fluorescence signal is 1.5. According to an embodiment of the present invention, the fourth droplet with a fluorescence signal of the second fluorescence signal less than 1.5 can be considered to contain the target bacteria, and the fourth droplets with a fluorescence value of the second fluorescence signal less than 1.5 are collected, so as to effectively screen out the target bacteria with antibacterial function from the mixed flora of the bacteria to be screened.

[0048] According to an embodiment of the present invention, the bacteria to be screened include Bacillus, and the indicator bacteria include Staphylococcus aureus.

[0049] According to an embodiment of the present invention, the fluorescent label includes at least one of GFP, RFP, and YFP.

[0050] According to some alternative embodiments of the present invention, the fluorescent label is GFP.

[0051] The solution of the present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0052] Example 1: High-throughput screening of antibacterial functional strains based on picoliter droplet system combined with fluorescence signal intensity. In this example, a microfluidic system is used to screen antibacterial functional strains. For the specific steps, see Figure 1 .

[0053] 1. Prepare the bacterial suspensions of the bacteria to be screened and the indicator bacteria

[0054] (1) Preparation of the bacterial suspension to be screened: Single colonies of Bacillus cereus (Bc) (Guyan, product number: GOYJ12378) and Escherichia coli (Ec) (China Center of Industrial Culture Collection, product number: CICC10899) were separately picked from the plate into LB medium, and cultured overnight at 37°C with shaking at 120 rpm. The next day, the bacterial suspensions of Bacillus cereus and Escherichia coli were centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and 5 mL of LB medium was added respectively to resuspend the cell pellets. The OD600 of the Bacillus cereus suspension was adjusted to 0.05 with LB medium, and the OD600 of the Escherichia coli suspension was adjusted to 0.03 with LB medium. 500 μL of Bacillus cereus with an OD600 of 0.05 and 500 μL of Escherichia coli with an OD600 of 0.03 were respectively pipetted into the injection vial and mixed well by pipetting (Bacillus cereus simulates the antibacterial strain group of Staphylococcus aureus in the environment, and Escherichia coli simulates the non-antibacterial strain group of Staphylococcus aureus in the environment).

[0055] (2) Preparation of the indicator bacterial suspension: The method described in step 1 of this example was used to culture Staphylococcus aureus with a green fluorescent label (the strain itself is Staphylococcus aureus 29213, from the laboratory of Teacher Zhu Kui of China Agricultural University, Sa), and the OD600 of the Staphylococcus aureus suspension was adjusted to 0.6 with LB medium. 1 mL of the Staphylococcus aureus suspension was pipetted into the injection vial.

[0056] 2. Generation of the first droplet

[0057] Take the bacterial suspension to be screened, insert it into the droplet generation chip, prepare about 1 million first droplets with a diameter of about 30 μm, and collect them in a Teflon tube. For the specific situation of the first droplet generation, see Figure 2 .

[0058] 3. Generation of the second droplet

[0059] Place the Teflon tube containing the first droplets collected in step 2 in fluorinated oil to prevent the drying of the first droplets, transfer it to a 37°C constant temperature incubator and let it stand for 2 h to generate the second droplets. For the bright-field microscopy image of the second droplets, see Figure 3 .

[0060] 4. Microscopic examination of the second droplets

[0061] Place the second droplets in an Automated Cell Counter, observe the integrity of the second droplets under the microscope, count the bacteria-carrying rate of the second droplets to estimate the single-cell rate, observe the growth of the bacteria to be screened in the second droplets, and control the integrity and uniformity of the second droplets.

[0062] 5. Microinjection of the indicator bacteria

[0063] Put the second droplet with qualified microscopy results and the suspension of the indicator bacteria (Staphylococcus aureus) to be microinjected onto the microinjection chip together, ensuring that each indicator bacterium is microinjected into a second droplet containing a bacterium to be screened, generating a third droplet. Collect about 500,000 third droplets in a Teflon tube. The process of microinjecting the indicator bacteria can be seen in Figure 4 .

[0064] 6. Quality control of the third droplet

[0065] Insert the third droplet with microinjected indicator bacteria into the droplet sorting chip, set the PMT to 3.8, and the fluorescence sorting range to 3.5 - 10. Collect the third droplets within this range and store them in a Teflon tube. The process of quality controlling the third droplet through the fluorescence sorting value can be seen in Figure 5 .

[0066] 7. Incubate the third droplet at a constant temperature

[0067] Put the Teflon tube containing the third droplets with a fluorescence sorting range of 3.5 - 10 into fluorinated oil to prevent the third droplets from drying. Transfer it to a constant temperature incubator at 37°C and let it stand for 18 hours to generate a fourth droplet, enabling the indicator bacteria and the bacteria to be screened to fully symbiose or antagonize in the fourth droplet. The fluorescence field microscopy image of the constant temperature incubation of the third droplet can be seen in Figure 6 .

[0068] 8. Microscopy after co - incubation

[0069] Put the fourth droplet after co - incubation into an Automated Cell Counter and observe the general situation of the indicator bacteria with fluorescent labels and the bacteria to be screened under a fluorescence microscope to quality control the integrity and uniformity of the fourth droplet.

[0070] 9. Sort the fourth droplet

[0071] Put the intact and uniform fourth droplet after quality control onto the fluorescence sorting chip for signal detection and data collection. Set the PMT to 3.2, collect the fourth droplets with a fluorescence signal less than 1.5, and the non - collected channels are the fourth droplets with a fluorescence signal greater than 1.5. Verify the results through a plate to prove that this method can effectively screen out the antibacterial strain group Bacillus from the mixed flora of Bacillus and Escherichia coli. The specific results can be seen in Figure 7 .

[0072] Among them, Figure 7 Sort refers to the fourth droplets with a fluorescence signal less than 1.5. The upper part is the microscopy image, and the lower part is the re - verification image of the plate. The plate can identify only Bacillus and a small amount of the indicator bacteria Staphylococcus aureus through colony morphology. Figure 7The Waste in it is the fourth droplet with a fluorescence signal greater than 1.5. The upper part is the microscopic examination image, and the lower part is the plate re-verification image. The plate can identify Escherichia coli, a small amount of spores, and a large amount of the indicator bacterium Staphylococcus aureus through colony morphology.

[0073] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0074] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for screening strains with antibacterial function, characterized in that, Including: Subjecting the bacteria to be screened and the indicator bacteria to screening treatment in a microfluidic system, wherein the indicator bacteria have fluorescent labels, so as to obtain target bacteria, and the target bacteria have the activity of inhibiting the indicator bacteria.

2. The method according to claim 1, characterized in that, The microfluidic system includes a droplet generation chip, a micro-injection chip and a droplet sorting chip; The screening treatment is carried out in the following manner: Subjecting the bacteria to be screened to a first droplet generation treatment to obtain first droplets, and each of the first droplets contains one strain of the bacteria to be screened; Subjecting the first droplets to a first culture treatment to obtain second droplets; Micro-injecting the indicator bacteria into the second droplets to obtain third droplets; Based on the first fluorescence signal of the third droplets, subjecting the third droplets to a second culture treatment to obtain fourth droplets; Based on the second fluorescence signal of the fourth droplets, obtaining target fourth droplets, and the target fourth droplets contain the target bacteria.

3. The method according to claim 2, characterized in that, Before carrying out the first droplet generation treatment, it further includes subjecting the bacteria to be screened to pre-culture treatment of the bacteria to be screened. After the pre-culture treatment of the bacteria to be screened, the OD600 of the bacteria to be screened is 0.03-0.

05.

4. The method according to claim 2, characterized in that, Before injecting the indicator bacteria into the second droplets, it further includes subjecting the indicator bacteria to pre-culture treatment. After the pre-culture treatment of the indicator bacteria, the OD600 of the indicator bacteria is 0.4-0.

8.

5. The method according to claim 4, characterized in that, Before injecting the indicator bacteria into the second droplets, it further includes subjecting the indicator bacteria to pre-culture treatment. After the pre-culture treatment of the indicator bacteria, the OD600 of the indicator bacteria is 0.

6.

6. The method according to claim 2, characterized in that, The first droplet generation treatment is carried out in the droplet generation chip.

7. The method according to claim 2, characterized in that, The diameter of the first droplet is 10 to 50 μm, and the generation rate of the first droplet is 10 6 to 10 8 droplets / h.

8. The method according to claim 2, characterized in that, The first culture treatment is carried out at a temperature of 35-42°C for 1-5 h.

9. The method according to claim 8, characterized in that, The first culture treatment is carried out at a temperature of 37°C for 2 h.

10. The method according to claim 2, characterized in that, The first culture treatment is carried out in the presence of at least one of fluorinated oil, mineral oil and silicone oil.

11. The method according to claim 2, characterized in that, Injecting the indicator bacteria into the second droplets is carried out in the micro-injection chip.

12. The method according to claim 2, characterized in that, The generation rate of the third droplet is 10 4 ~10 6 per hour.

13. The method according to claim 2, characterized in that, The first fluorescence signal of the third droplets is obtained in the droplet sorting chip.

14. The method according to claim 2, characterized in that, The fluorescence sorting range of the first fluorescence signal is 3.5-10.

15. The method according to claim 2, characterized in that, The second culture treatment is carried out at a temperature of 35-42°C for 15-20 h.

16. The method according to claim 15, characterized in that, The second culture treatment is carried out at a temperature of 37°C for 18 h.

17. The method according to claim 2, characterized in that, The second culture treatment is carried out in the presence of at least one of fluorinated oil, mineral oil and silicone oil.

18. The method according to claim 2, wherein, The generation rate of the fourth droplet is 10 4 ~10 6 per hour.

19. The method according to claim 2, wherein, The second fluorescence signal of the fourth droplets is obtained in the droplet sorting chip.

20. The method according to claim 2, wherein, The fluorescence sorting threshold of the second fluorescence signal is 1.

5.

21. The method according to claim 1, wherein, The bacteria to be screened include Bacillus, and the indicator bacteria include Staphylococcus aureus.

22. The method according to claim 1, wherein, The fluorescent label includes at least one of GFP, RFP and YFP.

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