Method for efficient targeted separation of bacteriophage based on modified medium and application
By surface activation and cross-linking treatment of modified cellulose sponge media, bacteria are fixed and then filtered into water samples, which solves the problems of low throughput and poor versatility of existing phage enrichment methods and achieves efficient and convenient phage separation.
Patent Information
- Application Number
- CN202510982746.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-28
AI Technical Summary
Existing phage enrichment methods suffer from low throughput and poor versatility. In particular, suspended particles and dissolved organic matter in water samples affect phage recovery efficiency, and membranes are prone to clogging, resulting in low filtration efficiency.
Modified cellulose sponge was used as a medium. The surface hydroxyl groups were activated to amino groups, and the samples were pretreated with cross-linking agent EDC. After the bacteria were fixed, the water samples were filtered to achieve targeted separation of bacteriophages.
It achieves high-throughput, low-cost, and easy-to-operate targeted phage separation, applicable to water bodies with different physicochemical properties, with a high capture success rate, and suitable for phage separation under different environments.
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Figure CN120843448A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application for efficient targeted separation of bacteriophages based on modified media. Background Technology
[0002] bacteriophage ( Bacteriaphage, phage Bacteriophages are the most numerous and specific viruses that infect prokaryotes such as bacteria and archaea. They are widely distributed in nature, including soil, air, water, plants, and the intestines of humans and animals. Bacteriophages exhibit diverse morphologies, including icosahedral, tailed, polyhedral, filamentous, and polygonal types. Based on their tail structure, bacteriophages are further divided into three families: Podoviridae, Siphoviridae, and Myoviridae, accounting for over 96% of known bacteriophages. With advancements in sequencing technology and the continuous enrichment of bacteriophage genome libraries, the ICTV abolished morphological classification of bacteriophages in 2022, establishing a more systematic classification system based on morphological and genomic characteristics, using order, family, genus, and species as the basic framework.
[0003] Currently, phage enrichment mainly employs methods such as ultrafiltration and sedimentation centrifugation to process water samples, and phages are recovered through a double-layer agar coating method.
[0004] Ultrafiltration involves applying positive pressure to a water sample and passing it through a filter membrane to remove large molecular impurities and some small molecular solutes. Finally, the ultrafiltration membrane is used to trap and enrich bacteriophage particles. This method has the advantages of not requiring sample acidification, simultaneously concentrating multiple bacteriophages, and not being limited by water sample volume. However, different water qualities affect the bacteriophage recovery efficiency. In particular, suspended particles and dissolved organic matter (DOMs) in the water sample are negatively charged, severely hindering the adsorption and elution of bacteriophages on the membrane surface. Furthermore, the membrane aperture is relatively small, making it prone to clogging during ultrafiltration, resulting in low filtration efficiency.
[0005] Precipitation centrifugation primarily uses precipitants such as zinc chloride to precipitate bacteriophage particles in environmental samples, and then concentrates the phage solution by centrifugation to increase the concentration. Compared to ultrafiltration, it is suitable for small-volume water samples and can separate bacteriophages in low-concentration phage solutions (10-100 PFU / mL), greatly improving the separation efficiency. However, its throughput is low, making it unsuitable for separating phages from large volumes of water, thus limiting its versatility.
[0006] Therefore, it is essential to develop a method that has high throughput, good versatility, and high targeted separation efficiency. Summary of the Invention
[0007] To address at least one of the aforementioned problems, this invention provides a method and application for efficient targeted separation of bacteriophages based on modified media.
[0008] To achieve the above objectives, the present invention employs the following technical means: The first aspect of the present invention provides a method for efficient targeted separation of bacteriophages based on modified media, characterized by comprising the following steps: S1. Using cellulose sponge as a medium, the medium is reacted with APTES: 3-aminopropyltriethoxysilane in an anhydrous reaction system constructed with an inert gas, and the surface activated hydroxyl groups are converted into amino groups. S2, terminate the reaction and remove APTES: 3-aminopropyltriethoxysilane; S3. Apply 1 mM of crosslinking agent EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the surface of the medium to complete the pretreatment and obtain the modified medium; S4. Use a coating stick to apply the bacterial cells to the surface of the modified medium for cross-linking; S5. Filter the water sample to be separated using the modified medium that has been treated previously.
[0009] In some embodiments of the present invention, the reaction conditions in step S1 are 80°C for 10-12 hours.
[0010] In some embodiments of the present invention, the cross-linking conditions between the bacterial cells and the modified medium in step S4 are 4°C and 25-30 min.
[0011] In some embodiments of the present invention, the bacterial cells mentioned in step S4 are bacteriophage host bacteria, and there are various choices of bacteriophage host bacteria, including but not limited to Citrobacter freundii, Klebsiella pneumoniae, etc.
[0012] In some embodiments of the present invention, the method further includes the step of removing the filtered medium and using a phage host bacterial culture to expand the culture and release the phage.
[0013] In some embodiments of the present invention, a step is also included to observe whether the phage has been successfully captured using the double-layer agar plate method.
[0014] A second aspect of the present invention provides a method for modifying phage media for efficient targeted separation, comprising the following steps: S1. Using cellulose sponge as a medium, react the medium with APTES in an anhydrous reaction system built with inert gas at 80℃ for 10-12 hours to convert the surface activated hydroxyl groups into amino groups. S2, terminate the reaction and remove APTES; S3. Apply 1mM crosslinking agent EDC to the surface of the medium to obtain the modified medium.
[0015] A third aspect of the present invention provides a modified medium, wherein the medium is a modified cellulose sponge, which is prepared by modification using the method described in the second aspect.
[0016] The fourth aspect of the present invention provides the application of the modified media described in the third aspect in the efficient targeted separation of bacteriophages.
[0017] The fifth aspect of the invention provides the use of the modified medium described in the third aspect in the preparation of products related to phage detection.
[0018] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a modified cellulose sponge as a medium, which is a polysaccharide material, similar to a sponge material, with a loose and porous structure, which can efficiently filter a large amount of water; it has good biocompatibility, and its surface is rich in hydroxyl groups, which has the basis for adsorbing and fixing bacteria. By modifying its surface through chemical methods, it can fix different types of strains, including Gram-positive bacteria and Gram-negative bacteria.
[0019] This invention is based on the biological characteristics of the specific binding between bacteria and bacteriophages. It fixes bacteria on a loose and porous medium to achieve targeted separation of bacteriophages. This method has the advantages of low cost, convenient operation, targeted separation, and high throughput. It is suitable for targeted separation of bacteriophages in water bodies with different physicochemical properties, and the success rate of bacteriophage capture is high. Attached Figure Description
[0020] Figure 1 The results of the evaluation of the phage targeting and capture effect of different media in Example 2 of the present invention are shown. Figure 2 The results of the double-layer agar plate method for detecting phages captured by the targeted separation method in Example 3 of the present invention are shown. Figure 3 The results of bacteriophage isolation from natural waters in Example 4 of the present invention are shown; Figure 4 The comparative genomic results of 10 bacteriophages isolated from natural waters using the targeted separation method in Example 4 of this invention are shown. Detailed Implementation
[0021] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.
[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.
[0023] The technical solution of this application will be further described in detail below with reference to specific embodiments.
[0024] Example 1: Media Modification and Immobilized Bacteria I. Medium Modification Three media were prepared: (1) cellulose sponge; (2) defatted cotton; (3) chitosan fiber. The media were pretreated to activate the hydroxyl groups. The operation steps are as follows: 1. Construct an anhydrous reaction system using inert gases; 2. The above three media were reacted with APTES (3-aminopropyltriethoxysilane) in an anhydrous reaction system at 80°C for 12 hours, and the surface activated hydroxyl groups were converted into amino groups. 3. Terminate the reaction and remove APTES; 4. Apply 1mM crosslinking agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) to the surface of the medium.
[0025] II. Bacterial Immobilization in Media Prepare two target bacteria: (1) FSNM: Citrobacter freundii; (2) Pae: Klebsiella pneumoniae; culture the target bacteria to the logarithmic phase and centrifuge to collect the bacterial cells.
[0026] The specific operation for fixing bacteria with a medium is as follows: the target bacteria are coated onto the surface of the modified medium using a coating stick, and the reaction is carried out at 4 ℃ for 30 min to activate the cross-linking of bacterial surface functional groups with the medium.
[0027] Then, the bacterial immobilization efficiency of the media after the cross-linking reaction was completed was evaluated. The evaluation process is as follows: 30 mL each of FSNM (Citrobacter freundii) and Pae (Klebsiella pneumoniae) bacteria were cultured overnight to the exponential growth phase. The cultures were thoroughly vortexed, and 100 μL were spread onto a plate. Then, three media—absorbent cotton, chitosan fibers, and cellulose sponges—were thoroughly immersed in the bacterial culture. The media were then removed and squeezed dry with tweezers. The remaining bacterial culture was thoroughly vortexed, and 100 μL was spread onto a plate. The removed media were then placed in 30 mL of sterile PBS buffer and incubated for 20 min at 200 rpm. -1The medium was eluted on a shaker, then the water was squeezed out, vortexed, and 100 μL was spread. The elution step was repeated 3 times, with the medium placed in fresh PBS buffer each time. The spread plate was incubated overnight at 28°C for counting.
[0028] The formula for counting bacteria fixed in the medium is: (original bacterial concentration - concentration of liquid applied in sequence) / original bacterial concentration × 100%. The solidification efficiency of the medium after 3 washes is the sum of the concentrations of the liquid applied in sequence and the difference between the concentrations of the liquid applied in sequence.
[0029] The solidification effect after adsorption and after elution once, twice and three times is shown in Table 1.
[0030] Table 1 Evaluation of bacterial fixation efficiency in the medium
[0031] The results showed that all three media had good solidification ability against both types of bacteria, and could maintain a solidification rate of over 95% even after multiple washes.
[0032] Example 2: Phage targeting and capture effect on different media The success rate of targeting and capturing bacteriophages using the three media described in Example 1 was evaluated.
[0033] Two treatments are set up for each medium: non-solidified medium and solidified medium.
[0034] The specific operation procedure for phage targeted capture is as follows: 1. Place bacteriophages of three different hosts, Pae, FSNM, and S1, in ddH2O to prepare bacteriophage water samples with a concentration of 10 PFU / L. 2. Filter the bacteriophage water samples using different media; 3. Remove the medium and place it in the target bacterial solution for amplification culture to release bacteriophages; 4. Use the double-layer agar plate method to observe whether the phage was successfully captured.
[0035] Evaluation results of the targeted capture and isolation effects of the three media on each type of phage are as follows: Figure 1 As shown.
[0036] The results showed that all three media could target and separate bacteriophages; among them, the cellulose sponge bacteriophage group successfully separated bacteriophages from three different hosts with a capture rate of 100%.
[0037] Example 3: Comparison of phage capture effects between targeted and non-targeted separation methods The phage solution used in this experiment was prepared by mixing the 10 types of phages listed in the table below in ddH2O.
[0038] Table 2 Composition of bacteriophage fluid
[0039] (a) The procedure for targeted isolation of bacteriophages is as follows: 1. The pretreated medium was reacted with APTES (3-aminopropyltriethoxysilane) in an anhydrous reaction system constructed with inert gas at 80°C for 12 hours to convert the surface activated hydroxyl groups to amino groups; after terminating the reaction, APTES was removed. 2. Apply 1 mM of crosslinking agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) to the surface of the medium; 3. Use a coating stick to apply the host bacteria to the surface of the modified medium, react at 4 ℃ for 30 min to activate the cross-linking of bacterial surface functional groups with the medium; 4. Use a medium with well-cross-linked bacterial cells to filter the bacteriophage fluid; 5. Remove the medium and place it in the target bacterial solution for amplification culture to release the bacteriophage; 6. Use the double-layer agar plate method to observe whether the phage was successfully captured.
[0040] (II) The procedure for non-targeted isolation of bacteriophages is as follows: 1. Screen the host bacteria corresponding to the bacteriophage, prepare the host bacterial solution, add flocculant to the host bacterial solution and let it stand overnight, then centrifuge at 5000 rpm for 50 min, discard the supernatant, and store the precipitate in SM buffer; 2. Select host bacteria and expand their culture to prepare host bacterial solution; 3. Add the phage fluid obtained from flocculation to the host bacterial culture and screen the phage using the agar plate method.
[0041] The experimental results using the above-mentioned targeted phage capture method and the non-targeted phage capture method are shown in Table 2 and... Figure 3 As shown.
[0042] Table 3. Phage capture efficiency of different methods
[0043] Note: + indicates successful capture of bacteriophage.
[0044] The results showed that the modified medium-based solid-state bacteria targeted phage separation method successfully captured phages from 5 different hosts, achieving a capture rate of 50%, while the non-targeted separation method only successfully isolated phages from 2 different hosts. The capture rate of the targeted separation method was significantly better than that of the non-targeted separation method. Furthermore, by… Figure 3 It can be seen that the targeted separation method captures phages with high abundance, which facilitates observation and subsequent phage isolation.
[0045] Example 4: Phage capture effect of targeted method in natural water bodies To evaluate the effectiveness of the phage targeted isolation method in natural environments, this embodiment used water samples collected from natural water bodies in Huangpu District, Guangzhou. Fifty bacterial strains (not 16S identified) were isolated from the water samples and purified and cultured for subsequent phage isolation. The isolation method was the same as the non-targeted isolation method for phage capture in Example 3 (II).
[0046] The results showed that phages were isolated from 11 bacterial strains, with a capture rate of 22%; subsequent titration experiments confirmed the presence of clear phage plaques, such as... Figure 4 As shown, this demonstrates that these bacteriophages have strong activity and lytic activity.
[0047] Simultaneously, using the targeted isolation method (I) in Example 3, non-native isolated strains—Citrobacter freundii and Klebsiella pneumoniae—were selected for targeted isolation, ultimately successfully capturing 10 bacteriophage strains. The genome detection results are as follows: Figure 4 As shown.
[0048] Therefore, the method for targeted phage capture based on modified media in this invention has the characteristics of high throughput and high targeted capture rate, and is applicable to the targeted separation of phages under different environments.
[0049] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.
Claims
1. A method for efficient targeted separation of bacteriophages based on modified media, characterized in that, The steps include: S1. Using cellulose sponge as a medium, the medium is reacted with 3-aminopropyltriethoxysilane in an anhydrous reaction system constructed with an inert gas, and the surface activated hydroxyl groups are converted into amino groups. S2, terminate the reaction and remove 3-aminopropyltriethoxysilane; S3. Apply 1 mM of crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the surface of the medium to complete the pretreatment and obtain the modified medium; S4. Use a coating stick to apply the bacterial cells to the surface of the modified medium for cross-linking; S5. The bacteriophage can be separated by filtering the water sample with the previously treated modified medium.
2. The method for efficient targeted separation of bacteriophages based on modified media according to claim 1, characterized in that, The reaction conditions in step S1 are 80℃ for 10-12 hours.
3. The method for efficient targeted separation of bacteriophages based on modified media according to claim 1, characterized in that, In step S4, the cross-linking conditions between the bacterial cells and the modified medium are 4℃ and 25-30 min.
4. The method for efficient targeted separation of bacteriophages based on modified media according to claim 1, characterized in that, The bacteria in question are bacteriophage host bacteria.
5. The method for efficient targeted separation of bacteriophages based on modified media according to claim 1, characterized in that, It also includes the step of removing the filtered medium and using a phage host bacterial culture to expand the culture and release the phage.
6. The method for efficient targeted separation of bacteriophages based on modified media according to claim 1, characterized in that, It also includes a step of using the double-layer agar plate method to observe whether the phage was successfully captured.
7. A method for modifying phage media for efficient targeted separation, characterized in that, The steps include: S1. Using cellulose sponge as a medium, react the medium with APTES in an anhydrous reaction system built with inert gas at 80℃ for 10-12 hours to convert the surface activated hydroxyl groups into amino groups. S2, terminate the reaction and remove APTES; S3. Apply 1mM crosslinking agent EDC to the surface of the medium to complete the pretreatment and obtain the modified medium.
8. A modified medium, characterized in that, The medium is a modified cellulose sponge, prepared by modification using the method described in claim 7.
9. The application of the modified medium according to claim 8 in the efficient targeted separation of bacteriophages.
10. The use of the modified medium according to claim 8 in the preparation of products related to phage detection.