JP-7856305-B2 - Methods for detecting microbial growth, methods for obtaining microorganisms, kits for detecting microbial growth, kits for obtaining microorganisms, and use of dyes as microbial growth reporters.

JP7856305B2JP 7856305 B2JP7856305 B2JP 7856305B2JP-7856305-B2

Inventors

佐々木章
大田悠里
野田尚宏
横田亜紀子
陶山哲志

Assignees

国立研究開発法人産業技術総合研究所

Dates

Publication Date: 20260511
Application Date: 20220729

Claims (6)

To prepare droplets containing microorganisms and styryl dye in a W/O emulsion, The microorganisms are cultured in the droplet, Based on the fluorescence intensity of the styryl dye , the growth of the microorganism is detected with a lower limit of 10 cells as the average number of cells per droplet . Includes , The aforementioned 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 Method for detecting microbial growth.
The aforementioned microorganisms There are multiple species. The method for detecting microbial growth according to claim 1 .
To prepare droplets containing microorganisms and styryl dye in a W/O emulsion, The microorganisms are cultured in the droplet, Based on the fluorescence intensity of the styryl dye , the growth of the microorganism is detected with a lower limit of 10 cells as the average number of cells per droplet . The droplets in which the growth of the aforementioned microorganisms was detected are collected, Includes , The aforementioned 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 How to obtain microorganisms.
The material comprises 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 , A kit for detecting microbial growth in droplets in a W/O emulsion , for use in the microbial growth detection method described in claim 1 or 2 .
The material comprises 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 , A kit for acquiring microorganisms in droplets in a W/O emulsion for use in the method for acquiring microorganisms described in claim 3 .
The use of styryl dye as a microbial growth reporter in droplets in a W/O emulsion , The lower limit of the average number of cells contained in one droplet detected by the microbial growth reporter is 10 cells. The aforementioned 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 use.

Description

This invention relates to a method for detecting microbial growth, a method for obtaining microorganisms, a kit for detecting microbial growth, a kit for obtaining microorganisms, and the use of a dye as a microbial growth reporter. A known method for isolating or culturing microorganisms is the use of water-in-oil (W/O) emulsions. In this method, droplets (culture media) are dispersed in the oil phase, and microorganisms are cultured in each droplet as a single culture medium. By using microfluidic channels to form these droplets, hundreds of thousands to millions of droplets can be formed in a few minutes, enabling high-throughput cultivation. Furthermore, by adjusting the number of microorganisms in the aqueous phase and the number of droplets, it is possible to form droplets containing a single microbial cell, enabling cultivation from a single cell. When culturing microorganisms using droplets, the number of cells per droplet follows a Poisson distribution. As the proportion of droplets containing only one cell increases, the probability of droplets lacking microorganisms also increases. In this case, selectively recovering droplets showing microbial growth can be useful for analyzing each microorganism and for scaling up the culture. Methods for detecting droplets containing microorganisms include detecting the autofluorescence of microbial cells and detecting them via reagents that react 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. This fluorescently modified nucleic acid probe has a fluorescent group and a quenching group at its 5' and 3' ends, respectively. Within a droplet containing microorganisms, the fluorescently modified nucleic acid probe is cleaved by RNase secreted by the microorganisms, resolving the FRET effect and increasing the fluorescence intensity. International Publication No. 2019/073902 This figure shows a microscopic image of the droplet after culture in Test Example 1. The bars in the figure represent 100 μm.This figure shows the fluorescence intensity of droplets in Test Example 2. (A) shows the fluorescence intensity of droplets without cell membrane staining reagent. (B) and (C) show the fluorescence intensity of droplets containing cell membrane staining reagent.This figure shows a microscopic image of the droplet after culture in Test Example 3. The bars in the figure represent 100 μm.This figure shows the fluorescence intensity of droplets containing a dye that emits green fluorescence in Test Example 4.This figure shows the fluorescence intensity of droplets containing a red fluorescent dye in Test Example 4. Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments described below. (Method for detecting microbial growth) The microbial growth detection method according to this embodiment includes: (Step 1) preparing droplets containing a dye whose fluorescence intensity changes upon interaction with microorganisms and membrane components of the microorganisms in a W/O emulsion; (Step 2) culturing microorganisms in the droplets; and (Step 3) detecting microbial growth based on the fluorescence intensity of the dye. A W/O emulsion refers to a state in which particulate droplets (water droplets) exist as a dispersed phase within a continuous oil phase. A "droplet" refers to a compartmentalized water droplet within the emulsion. The aqueous phase constituting the W/O emulsion is not particularly limited, as long as it is a hydrophilic liquid that does not mix with the oil phase. Suitable liquids for the aqueous phase include, for example, water, lake water, and seawater. However, since microorganisms are cultured within the droplets, a culture medium that does not mix with the oil phase, such as LB medium and R2A medium, is preferred. The aqueous phase for the droplet prepared in Step 1 contains microorganisms and the above-mentioned dye. The microorganisms are not particularly limited as long as they are capable of growing within the droplet. The microorganisms may be prokaryotes or eukaryotes. Examples of microorganisms include E. coli, Bacillus subtilis, actinomycetes, and yeast. The microbial growth detection method according to this 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 present in the environmental sample, i.e., multiple species of microorganisms. Furthermore, the microorganisms may be artificially created microorganisms, such as genetically modified microorganisms. The dye is one whose fluorescence intensity changes, preferably increases, by interacting with membrane components of microorganisms within the droplet. Preferably, the dye is one whose fluorescence inte