Method for detecting growth of microorganisms, method for obtaining microorganisms, kit for detecting growth of microorganisms, kit for obtaining microorganisms, and use of dye as microbial growth reporter

Abstract

To provide a method for detecting the growth of microorganisms that can accurately detect a droplet in which microorganisms have grown, to provide a method for obtaining microorganisms, to provide a kit for detecting the growth of microorganisms, to provide a kit for obtaining microorganisms, and to provide use of dye as microbial growth reporter.SOLUTION: A method for detecting the growth of microorganisms comprises: producing a droplet containing a dye whose fluorescence intensity changes by interacting with a microorganism and a membrane component possessed by the microorganism, in W / O emulsion; culturing the microorganism within the droplet; and detecting the growth of the microorganism based on the fluorescence intensity of the dye.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for detecting microbial growth, a method for obtaining a microorganism, a kit for detecting microbial growth, a kit for obtaining a microorganism, and the use of a dye as a microbial growth reporter. [Background technology]

[0002] A method using water-in-oil (W / O) emulsion is known as a method for isolating or culturing microorganisms. In this method, droplets (culture medium) are dispersed in an oil phase, and each droplet is used as a culture site to culture microorganisms. By using a microchannel in the droplet formation method, hundreds of thousands to millions of droplets can be formed in a few minutes, making it possible to achieve high throughput. In addition, by adjusting the number of microorganisms and droplets present in the aqueous phase, droplets containing one microbial cell can be formed, making it possible to culture from a single cell.

[0003] When droplets are used to culture microorganisms, the number of cells per droplet follows a Poisson distribution. As the ratio of droplets containing only one cell increases, the probability of droplets containing no microorganisms also increases. In this case, if droplets showing microbial growth can be selectively collected, they can be used for analysis of each microorganism and scale-up.

[0004] Droplets containing microorganisms are detected by a method of detecting the autofluorescence of microbial cells and a method of detecting via a reagent that reacts with extracellular secretions. For example, Patent Document 1 discloses the use of a fluorescence resonance energy transfer (FRET) type fluorescently modified nucleic acid probe for detecting droplets containing microorganisms. The fluorescently modified nucleic acid probe has a fluorescent group and a quenching group at the 5' end and the 3' end, respectively. When the fluorescently modified nucleic acid probe is cleaved by RNase secreted from the microorganism in the droplet containing the microorganism, FRET is eliminated and the fluorescence intensity increases. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 073902 Summary of the Invention [Problem to be solved by the invention]

[0006] In the method of detecting the autofluorescence of cells, the sensitivity is low, so it is difficult to accurately detect droplets in which microorganisms have proliferated. In the method using a fluorescently modified nucleic acid probe disclosed in the above Patent Document 1, when attempting to detect microorganisms in an environmental sample, RNase contained in impurities in the environment and traces of RNase contained in the culture medium increase the background fluorescence value, resulting in false positives, and the proliferation of microorganisms in the droplets may not be accurately detected. In addition, there is a time lag between the actual proliferation and decomposition by RNase, and there is also the inconvenience that the number of cells present in the droplet is not directly correlated with the fluorescence intensity.

[0007] The present invention has been made in consideration of the above-mentioned situation, and aims to provide a microbial growth detection method, a microbial acquisition method, a microbial growth detection kit, a microbial acquisition kit, and use of a dye as a microbial growth reporter, which are capable of accurately detecting droplets in which microorganisms have grown. [Means for solving the problem]

[0008] The method for detecting microbial proliferation according to the first aspect of the present invention comprises: preparing droplets in a W / O emulsion, the droplets including a dye whose fluorescence intensity changes upon interaction with a microorganism and a membrane component of the microorganism; Cultivating the microorganism within the droplet; detecting growth of the microorganism based on the fluorescence intensity of the dye; Includes.

[0009] The dye is A dye whose fluorescence intensity increases when inserted into the outer membrane of a cell of the microorganism. This may also be the case.

[0010] The dye is N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide or 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrien-1-yl]-1-[3-(triethylammonio)propyl]-pyridinium dibromide. This may also be the case.

[0011] The microorganism is There are multiple species, This may also be the case.

[0012] The method for obtaining a microorganism according to the second aspect of the present invention comprises the steps of: preparing droplets in a W / O emulsion, the droplets including a dye whose fluorescence intensity changes upon interaction with a microorganism and a membrane component of the microorganism; Cultivating the microorganism within the droplet; detecting growth of the microorganism based on the fluorescence intensity of the dye; Recovering droplets in which growth of the microorganisms is detected; and Includes.

[0013] A kit for detecting microbial proliferation in droplets in a W / O emulsion according to a third aspect of the present invention comprises: The probe is equipped with a dye whose fluorescence intensity changes upon interaction with membrane components of microorganisms.

[0014] A kit for obtaining microorganisms in droplets in a W / O emulsion according to a fourth aspect of the present invention comprises: The probe is equipped with a dye whose fluorescence intensity changes upon interaction with membrane components of microorganisms.

[0015] Use of the dye according to the fifth aspect of the present invention as a microbial growth reporter in droplets in a W / O emulsion comprises: A dye whose fluorescence intensity changes upon interaction with membrane components of microorganisms is used as a reporter for microbial growth within droplets in a W / O emulsion. Effect of the Invention

[0016] According to the present invention, droplets in which microorganisms have proliferated can be detected with high accuracy. [Brief description of the drawings]

[0017] [Figure 1] 1 is a diagram showing a microscope image of droplets after culture in Test Example 1. The bar in the figure corresponds to 100 μm. [Diagram 2] 1 shows the fluorescence intensity of droplets in Test Example 2. (A) shows the fluorescence intensity of a droplet that does not contain a cell membrane staining reagent, (B) and (C) show the fluorescence intensity of droplets that contain a cell membrane staining reagent. [Diagram 3] 1 is a diagram showing a microscope image of droplets after culture in Test Example 3. The bar in the figure corresponds to 100 μm. [Figure 4] FIG. 13 is a graph showing the fluorescence intensity of droplets containing a dye that emits green fluorescence in Test Example 4. [Diagram 5] FIG. 13 is a graph showing the fluorescence intensity of droplets containing a dye that emits red fluorescence in Test Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described with reference to the accompanying drawings, in which: FIG.

[0019] (Microbial growth detection method) The microbial growth detection method according to this embodiment includes the steps of preparing a droplet in a W / O emulsion containing a microorganism and a dye whose fluorescence intensity changes upon interaction with a membrane component of the microorganism (step 1), culturing the microorganism within the droplet (step 2), and detecting the growth of the microorganism based on the fluorescence intensity of the dye (step 3).

[0020] A W / O emulsion is a state in which fine droplets (water droplets) exist as a dispersed phase in an oil phase, which is a continuous phase. The term "droplets" refers to compartmentalized water droplets in an emulsion. The aqueous phase constituting a W / O emulsion is not particularly limited as long as it is a hydrophilic liquid that is not miscible with the oil phase. Examples of liquids that can be used for the aqueous phase include water, lake water, and seawater, but in order to culture microorganisms in the droplets, a medium that is not miscible with the oil phase, such as LB medium and R2A medium, is preferable.

[0021] The aqueous phase for the droplets prepared in step 1 contains microorganisms and the above-mentioned dye. The microorganisms are not particularly limited as long as they can grow in the droplets. The microorganisms may be prokaryotic or eukaryotic. Examples of the microorganisms include Escherichia coli, Bacillus subtilis, actinomycetes, and yeast.

[0022] The microbial proliferation detection method according to the present embodiment can be applied to environmental samples such as air, lake water, seawater, and soil. Therefore, the microorganisms contained in the droplet may be microorganisms contained in the environmental sample, i.e., multiple types of microorganisms. Furthermore, the microorganisms may be artificially produced microorganisms, such as recombinant microorganisms into which genes have been introduced.

[0023] The dye is a dye whose fluorescence intensity changes, preferably increases, by interacting with a membrane component of the microorganism in the droplet. Preferably, the dye is a dye whose fluorescence intensity increases by being inserted into the outer membrane of the cell of the microorganism. Such dyes include styryl dyes. Styryl dyes are also called Fei Mao dyes, and examples of such dyes include N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide (FM (trademark, the same applies below) 1-43), 4-[2-[4-(dipentylamino)phenyl]ethenyl]-1-[3-(triethylaminio)propyl]pyridinium bromide (4-[2-[4-(Dipentylamino)phenyl]ethenyl]-1-[3-(triethylaminio)propyl]pyridinium bromide), and the like. bromide; FM1-84), N-(3-triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide; FM2-10), 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrien-1-yl]-1-[3-(triethylammonio)propyl]-pyridinium dibromide dibromide, FM4-64) and N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide, FM5-95).In particular, the dyes N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide and 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrien-1-yl]-1-[3-(triethylammonio)propyl]-pyridinium dibromide are preferred.

[0024] The oil phase constituting the W / O emulsion is not particularly limited as long as it is a hydrophobic liquid that is not miscible with the water phase. Examples of such oil phases include FC40, Novec (trademark, the same applies below) 7500, mineral oil, and combinations thereof.

[0025] To stabilize the W / O emulsion, a surfactant is added to at least one of the aqueous phase and the oil phase. Examples of surfactants used include Span (trademark) 80 and Tween (trademark) 20 for the aqueous phase. Examples of surfactants used include 008-FluoroSurfactant, Pico-surf (trademark) 1 and Krytox (trademark) for the oil phase. The concentration of the surfactant added to the aqueous phase or the oil phase can be appropriately adjusted depending on conditions such as the type of surfactant used and the size of the droplets.

[0026] The W / O emulsion may be any W / O emulsion as long as microorganisms can grow in the droplets and the growth of the microorganisms can be detected. The size of the droplets is not particularly limited as long as microorganisms can grow in the droplets and the growth of the microorganisms can be detected. The diameter of the droplets is, for example, 5 to 500 μm, 10 to 300 μm, 15 to 200 μm, or 20 to 150 μm.

[0027] Methods for preparing W / O emulsions are known, and they can be prepared using commercially available devices such as On-chip Droplet Generator (manufactured by On-chip Biotechnologies), Droplet Generator (manufactured by Bio-Rad), and QX100 (manufactured by Bio-RAD).

[0028] In step 2, a method for culturing microorganisms in droplets in a W / O emulsion is known, and the microorganisms can be cultured by appropriately selecting an apparatus required for culturing, such as a microchannel. The culture conditions may be any conditions that allow the microorganisms contained in the droplets to grow, and suitable culture conditions can be set according to the purpose of the culture or the microorganisms to be cultured. The temperature condition for the culture is, for example, 4 to 95°C. The culture time may be, for example, several hours, several days, or several months, or may be several days to several months or more.

[0029] In step 3, the proliferation of microorganisms is detected based on the fluorescence intensity of the dye. The fluorescence intensity of the dye can be measured using a commercially available device. Since the fluorescence intensity of the dye changes when it interacts with the membrane components of the microorganisms, the change in the fluorescence intensity can be detected by comparing it with the fluorescence intensity of the droplet before the start of cultivation or the droplet without microorganisms. When using a dye whose fluorescence intensity increases when it interacts with the membrane components of the microorganisms, as the number of microorganisms increases due to proliferation, the amount of the dye exposed to the membrane components increases, and the amount of the dye interacting with the membrane components increases. As a result, the fluorescence intensity increases in proportion to the number of microbial cells in the droplet.

[0030] The microbial proliferation detection method according to the present embodiment detects the proliferation of microorganisms based on the fluorescence intensity of a dye whose fluorescence intensity changes upon interaction with a membrane component possessed by the microorganism. This change in fluorescence intensity can be measured with higher sensitivity than that of cells to which the dye is not applied, so droplets in which microorganisms have proliferated can be detected with high accuracy. In addition, the microbial proliferation detection method according to the present embodiment detects the change in fluorescence intensity due to an increase in the dye that interacts with the membrane component, so that it is less susceptible to the influence of components other than the microorganisms contained in the environmental sample and the culture medium, and an increase in the background measurement value can be avoided. This allows the proliferation of microorganisms to be detected accurately. In addition, the change in fluorescence intensity due to an increase in the dye that interacts with the membrane component directly correlates with the actual proliferation of the microorganisms, so that the proliferation of the microorganisms can be detected without a time lag.

[0031] According to the microbial growth detection method of the present embodiment, by encapsulating and culturing multiple types of microorganisms in a single droplet, it is possible to verify the effect of interactions between the microorganisms encapsulated in the droplet on their growth ability.

[0032] Moreover, droplets containing microorganisms exhibiting different proliferation abilities can be obtained by encapsulating one cell of a microorganism per droplet. In particular, when the purpose is to isolate a specific microorganism from an environmental sample containing multiple types of microorganisms, it is preferable that each droplet contains one cell of the microorganism. Note that a method for producing a W / O emulsion so that each droplet contains one cell of the microorganism is known. For example, droplets containing one cell of the microorganism can be produced by adjusting the number of microorganisms and the number of droplets present in the aqueous phase according to the Poisson distribution.

[0033] In addition, when multiple cells are encapsulated in each droplet, it is preferable in that the culture time required for proliferation to a detectable cell number can be reduced. In addition, when symbiotic microorganisms are encapsulated in the same droplet, it is expected that the proliferation will be promoted by the interaction between the microorganisms.

[0034] Step 3 may be performed on a microchannel. By combining a microchannel with a commercially available cell sorter or the like, the fluorescence intensity can be measured with high throughput.

[0035] (How to obtain microorganisms) The method for obtaining microorganisms according to this embodiment includes, in addition to the above-mentioned steps 1, 2, and 3, recovering droplets in which microbial growth has been detected (step 4). Methods for recovering or separating droplets in which microbial growth has been detected, i.e., droplets in which the fluorescence intensity of the dye has changed before and after culture, are well known. For example, droplets in which the fluorescence intensity has changed can be sorted using a commercially available chip-type cell sorter. When step 4 is performed on a microchannel, the fluorescence intensity can be measured with high throughput by combining the microchannel with a commercially available cell sorter, and droplets can be separated according to the detection results.

[0036] According to the microorganism acquisition method of the present embodiment, droplets in which microorganisms have proliferated can be detected with high accuracy, so that desired microorganisms can be acquired more reliably. In addition, for environmental samples containing multiple types of microorganisms, by encapsulating one cell of a microorganism per droplet, proliferation according to the properties of each microorganism contained in each droplet can be detected, and droplets showing different proliferation capabilities can be isolated or separated. By selectively separating droplets showing different proliferation capabilities, analysis and culture of each microorganism can be scaled up.

[0037] (Microbial growth detection kit and microorganism acquisition kit) In another embodiment, a kit for detecting microbial proliferation in droplets in a W / O emulsion is provided. The kit for detecting microbial proliferation comprises the dye described above. The kit for detecting microbial proliferation may further comprise an aqueous phase of the W / O emulsion such as a culture medium, an oil phase of the W / O emulsion, and a surfactant for stabilizing the W / O emulsion. The kit for detecting microbial proliferation may comprise a microchannel and a culture dish for use in culturing, etc. In another embodiment, the kit for detecting microbial proliferation is used as a kit for obtaining microorganisms in droplets in a W / O emulsion.

[0038] (Microbial Growth Reporter) In another embodiment, the dye is used as a microbial growth reporter in a droplet in a W / O emulsion. The microbial growth reporter is an indicator of microbial growth in a droplet. When a dye whose fluorescence intensity increases when inserted into the outer membrane of a cell of a microorganism is used as the dye, the fluorescence intensity of the dye in the droplet correlates with the number of microbial cells in the droplet. EXAMPLES

[0039] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0040] (Test Example 1: Detection of E. coli in W / O droplets) We investigated whether the proliferation of Escherichia coli (E. coli) in W / O droplets could be detected by measuring the fluorescence of a cell membrane staining reagent. The following samples 1 to 3 were used as the aqueous phase. Sample 1: LB medium containing E. coli Sample 2: LB medium containing 10 μM cell membrane staining reagent FM1-43 (manufactured by ThermoFisher Scientific) and E. coli Sample 3: LB medium containing 10 μM cell membrane staining reagent FM4-64 (ThermoFisher Scientific) and E. coli

[0041] Novec7500 (manufactured by On-Chip Biotechnologies) containing 2% 008-FluoroSurfactant was used as the oil phase. Droplets with a diameter of about 30 μm were produced using an On-chip Droplet Generator as a W / O droplet production device. The concentration of E. coli in the aqueous phase was adjusted so that the average number of cells contained per droplet was 0.1 cells (0.1 cells / droplet). E. coli was statically cultured overnight at 37°C in the produced droplets.

[0042] (result) When the droplets after cultivation were observed under a microscope, it was observed that the droplets in which E. coli had grown showed strong fluorescence in Sample 2 and Sample 3, which contained the cell membrane staining reagent, as shown in Figure 1. This demonstrated that the growth of E. coli in W / O droplets can be detected by measuring the fluorescence of the cell membrane staining reagent.

[0043] (Test Example 2: Examination of the number of E. coli cells that can be detected using cell membrane staining reagent) The intensity of the cell membrane staining reagent was compared with the intensity of the autofluorescence to examine whether the detection sensitivity could be improved by using the cell membrane staining reagent. The following samples 4 to 9 were used as the aqueous phase. Sample 4: LB medium Sample 5: LB medium containing E. coli Sample 6: LB medium containing 10 μM FM1-43 Sample 7: LB medium containing 10 μM FM1-43 and E. coli Sample 8: LB medium containing 10 μM FM4-64 Sample 9: LB medium containing 10 μM FM4-64 and E. coli

[0044] Novec7500 containing 2% 008-FluoroSurfactant was used as the oil phase, and droplets with a diameter of about 30 μm were prepared in the same manner as in Test Example 1. The concentrations of E. coli in Samples 5, 7, and 9 were 10 cells / droplet, 50 cells / droplet, and 100 cells / droplet. The prepared droplets were statically cultured overnight at 37° C. Immediately after the droplets were prepared, the fluorescence intensity was measured using On-chip Sort (manufactured by On-chip Biotechnologies).

[0045] (result) As shown in Figure 2(A), in droplets that did not contain a cell membrane staining reagent, the difference in the number of E. coli cells could not be distinguished by the fluorescence intensity due to autofluorescence. On the other hand, in droplets that contained FM1-43 and FM4-64, as shown in Figure 2(B) and Figure 2(C), respectively, the fluorescence intensity increased according to the number of E. coli cells, and an increase in fluorescence intensity according to the number of cells was detected in droplets containing 10 or more E. coli cells.

[0046] (Test Example 3: Detection of environmental microorganisms using cell membrane staining reagent) We investigated whether cell proliferation could be detected when various types of microorganisms contained in soil were cultured using a cell membrane staining reagent. As the aqueous phase, LB medium containing 10 μM FM1-43 and soil microorganisms, and LB medium containing 10 μM FM4-64 and soil microorganisms were used. As the oil phase, Novec7500 containing 2% 008-FluoroSurfactant was used. Droplets with a diameter of approximately 100 μm were created using an On-chip Droplet Generator as a water-in-oil droplet creation device. Soil microorganisms were statically cultured in the created droplets overnight at 25°C.

[0047] (result) Images of the droplets observed under a microscope are shown in Figure 3. In both samples, it was observed under the microscope that the droplets in which soil microorganisms had proliferated showed increased fluorescence.

[0048] (Test Example 4: Detection of oleaginous yeast using cell membrane staining reagent) We investigated whether the cell proliferation of eukaryotic oleaginous yeast (Lipomyces starkeyi) could be detected. As the aqueous phase, a PBS suspension containing oleaginous yeast (100 cells / droplet) and various staining reagents (FM1-43 10 μM (referred to as dye #1), Cell Navigator (trademark, the same below) Green (manufactured by AAT) 500-fold diluted (referred to as dye #2), PlasMem Bright Green (manufactured by Dojindo) 200-fold diluted (referred to as dye #3), FM4-64 10 μM (referred to as dye #4), CellMask (trademark, the same below) Orange (manufactured by Invitrogen) 1000-fold diluted (referred to as dye #5), MemGlow (trademark, the same below) 560 (manufactured by Funakoshi) 20 μM (referred to as dye #6)) was used. For comparison, a sample without oleaginous yeast was used. Dye #6 is a dye whose fluorescence is minimized due to the formation of self-quenching nanoparticles before binding to the lipid bilayer membrane, but whose fluorescence intensity increases upon interaction with the lipid bilayer membrane.

[0049] Novec7500 containing 2% 008-FluoroSurfactant was used as the oil phase. Droplets with a diameter of about 120 μm were produced using a Droplet Generator as a water-in-oil droplet production device. Immediately after droplet production, the fluorescence intensity of each droplet was measured using On-chip Sort.

[0050] (result) 4 and 5 show the results for dyes that emit green fluorescence and dyes that emit red fluorescence, respectively. In droplets containing FM1-43 (dye #1) and FM4-64 (dye #4), the fluorescence intensity increased in response to the presence of oleaginous yeast. On the other hand, in the case of the other dyes #2, #3, #5, and #6, the staining intensity itself in the droplets was insufficient, or the background was too high, making it impossible to clearly distinguish the presence of oleaginous yeast.

[0051] The above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not by the embodiment. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0052] The present invention is suitable for testing for the presence or absence of microorganisms and for separating and analyzing microorganisms.

Claims

1. Producing droplets containing a microorganism and a styryl dye in a W / O emulsion; Cultivating the microorganism within the droplet; detecting the growth of the microorganism based on the fluorescence intensity of the styryl dye; A method for detecting microbial growth comprising:

2. The styryl dye is N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide, 4-[2-[4-(dipentylamino)phenyl]ethenyl]-1-[3-(triethylammonio)propyl]pyridinium bromide, N-(3-triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide, 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrien-1-yl]-1-[3-(triethylammonio)propyl]pyridinium dibromide or N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide, The method for detecting microbial growth according to claim 1.

3. The styryl dye is N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide or 4-[6-[4-(diethylamino)phenyl]-1,3,5-hexatrien-1-yl]-1-[3-(triethylammonio)propyl]-pyridinium dibromide, The method for detecting microbial growth according to claim 2.

4. The microorganism is There are multiple species, A method for detecting microbial growth according to any one of claims 1 to 3.

5. Producing droplets containing a microorganism and a styryl dye in a W / O emulsion; Cultivating the microorganism within the droplet; detecting the growth of the microorganism based on the fluorescence intensity of the styryl dye; Recovering droplets in which growth of the microorganisms is detected; and A method for obtaining a microorganism comprising the steps of:

6. A compound comprising a styryl dye. A kit for detecting microbial growth within droplets in a W / O emulsion.

7. A compound comprising a styryl dye. A kit for obtaining microorganisms in droplets in a W / O emulsion.

8. Use of a styryl dye as a microbial growth reporter within droplets in a W / O emulsion.

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