Method for efficiently enriching pig manure sample phage particles

Abstract

The invention discloses a method for efficiently enriching pig manure sample phage particles. The method comprises the following steps: fully dissolving a pig manure sample in an SM buffer solution, namely 100 mM of NaCl, 8 mM of MgSO4 and 50 mM of Tris with the pH value of 7.5, carrying out preliminary centrifugation, and collecting a supernatant; filtering the supernate by using a microfiltration membrane with the thickness of 0.22 mu m; collecting the filtrate, and adding PEG 8000 with the final concentration mass percent of 20% and NaCl with the final concentration mass percent of 0.6%; standing at 4 DEG C for more than 1 hour; centrifuging at 4 DEG C to precipitate PEG and virus particles; discarding the supernatant, and resuspending the sediment sample substance by using an SM buffer solution; repeating the steps of centrifugation, PEG and NaCl treatment and SM buffer solution resuspension; trichloromethane with the final concentration of 20% is added, and incubation is conducted for 10 min at the temperature of 20-25 DEG C; and then centrifugally separating a water phase, and transferring the water phase into a 1.5 mL centrifugal tube to obtain the water phase, namely a high-concentration virion concentrated solution. The method does not need an ultra-high-speed centrifuge, the reagent preparation method is simple, and the difficulty of reproduction of the experimental method is greatly reduced.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for enriching bacteriophage particles from pig feces samples and an application thereof. Background Art

[0002] Bacteriophages are the most abundant biological entities on Earth, with an estimated population of 10 31 In addition to the marine environment and soil, bacteriophages are also abundant in the mammalian intestine, accounting for about 97.9% of the total intestinal viruses. Bacteriophages play a key role in regulating the intestinal microbial ecosystem through phage predation, lysogeny, and horizontal gene transfer. Increasing evidence shows that bacteriophages have good application prospects in anti-infective treatment, recombinant antibody development, and biocontrol in the food industry.

[0003] In the past decade, it has been widely recognized that gut microbiota dysbiosis plays an important role in the development of many diseases. As pigs can provide meat for humans and serve as an important biomedical model, more and more research has focused on the pig gut microbiome and virome.

[0004] Pig feces samples contain a large number of bacteriophages. The present invention uses polyethylene glycol (PEG) to enrich and purify pig feces bacteriophages, and uses relevant biological monitoring technology to analyze and quantify the enriched bacteriophages. This is of great significance for bacteriophages as a new type of microecological preparation, which is used to regulate animal intestinal flora imbalance, improve animal body metabolism, and promote the healthy development of animal husbandry and breeding industry. Summary of the invention

[0005] The purpose of the present invention is to provide a method for enriching phage particles in pig feces samples.

[0006] To achieve the purpose of the present invention, the technical solution of the present invention is as follows: A method for efficiently enriching phage particles from pig fecal samples, wherein the pig fecal samples are fully dissolved in SM solution (100 mM NaCl, 8 mMM MgSO4, 50 mMTris [pH 7.5]), preliminarily centrifuged, and the supernatant is collected. The supernatant obtained after the preliminarily pretreatment is filtered with a 0.22 μm microporous filter membrane. The filtrate is collected and PEG 8000 and Nacl are added at a final concentration of 20% and 0.6%. The mixture is allowed to stand at 4°C for more than 1 hour. The mixture is then centrifuged at 4°C to precipitate PEG and virus particles. The supernatant is discarded and the precipitated material is resuspended in an appropriate amount of SM buffer. The above centrifugation, PEG, NaCl treatment, and SM resuspension steps are repeated. Chloroform (CHCl3) is added at a final concentration of 20%, and the mixture is incubated at room temperature (20°C-25°C) for 10 min. The aqueous phase is then separated by centrifugation and transferred to a new 1.5 mL centrifuge tube. The resulting aqueous phase is a high-concentration virus particle concentrate.

[0007] A method for efficiently enriching phage particles in pig feces samples comprises the following steps: 1. Enrichment of phage particles from pig feces samples (1.1) First, add sterile zirconium beads and 1 mL of SM buffer to a centrifuge tube containing 1.0 g of feces, then vortex for 5 minutes using a high-speed grinder, and then transfer to a 15 mL centrifuge tube. Then add SM buffer to 10 mL; (1.2) Centrifuge the sample at 6600 rpm and 4 °C for 30 min using a high-speed refrigerated centrifuge; (1.3) After centrifugation, take the supernatant and transfer it to a new centrifuge tube. Continue centrifugation in a high-speed refrigerated centrifuge (16,000 × g, 4 °C, 10 min). (1.4) Repeat step (3) 3 to 4 times until there is no precipitation in the sample; (1.5) After completion, transfer the supernatant to a new sterile 50 ml centrifuge tube, filter through 0.45 μm and 0.22 μm microporous filter membranes in sequence, and collect the filtrate into a new centrifuge tube; (1.6) Add 5X PEG8000 NaCl to the filtrate so that the volume ratio of 5X PEG8000 NaCl to the filtrate is 1:4; (1.7) After the sample is allowed to stand at 4°C for 1 hour (or overnight), it is centrifuged at 3400 g and 4°C for 60 minutes using a high-speed refrigerated centrifuge. (1.8) After centrifugation, discard the supernatant and resuspend the precipitate with an appropriate amount of SM buffer; (1.9) Repeat steps (1.6), (1.7) and (1.8); (1.10) Add CHCl3 to the filtrate to make the volume ratio of CHCl3 to filtrate 1:4; incubate at room temperature (20℃~25℃) for 10 min to remove the remaining bacteria, porcine intestinal cell fragments and polyethylene glycol (PEG). Transfer the aqueous phase obtained by centrifugation to a new 1.5mL centrifuge tube. The aqueous phase is the high-concentration phage particle concentrate; (1.11) Use a high-speed refrigerated centrifuge to centrifuge the sample at 5000g, 4°C for 15 min. The aqueous phase obtained by centrifugation is the high-concentration virus particle concentrate.

[0008] 2. Counting of phages after enrichment (2.1) Dilute SYBR™ Gold nucleic acid gel stain (10,000X concentrate) to 100X using dimethyl sulfoxide (DMSO). (2.2) Repeat steps (1.1) to (1.9) and add SYBR™ Gold stain to the separated concentrate so that the ratio of the diluted stain volume to the concentrate volume is 1:z99; (2.3) Incubate at 37°C in the dark for 1 hour or at 4°C overnight; (2.4) Add 5X PEG8000 NaCl to make the volume ratio of 5X PEG8000 NaCl to filtrate 1:4. Centrifuge at 23000g for 20 min at 4°C. Discard the supernatant and resuspend in SM buffer; (2.5) Repeat the above (2.4) to wash away excess dye to avoid false positive signals (2.6) The stained virus particle concentrate was diluted with SM buffer at 10, 100 and 1000 times gradient. When the flow cytometer was used for detection, the lateral angle SSC-H and channel FLl-H were selected for detection. Before the measurement, the flow cytometer automatically cleaned the inside to make the pipes unobstructed and the nozzles clean. The stained samples were loaded to count the number of positive phage particles at different gradient concentrations, and the number of phages isolated from each gram of pig feces was converted.

[0009] Furthermore, the preparation method of the SM buffer in the present invention is as follows: 2.0 g of MgSO4·7H2O, 0.1 g of gelatin, 5.8 g of NaCl and 50 mL of 1M Tris-HCl (pH=7.5) are mixed and the volume is adjusted to 100 mL with double distilled water. After high temperature and high pressure sterilization, the SM buffer is cooled and stored at 4°C; Furthermore, the preparation method of 5X PEG8000 NaCl is as follows: PEG8000 250g and NaCl 43.83g are mixed, and the volume is adjusted to 100mL with double distilled water. After high temperature and high pressure sterilization, the 5X PEG8000 NaCl solution is cooled and stored at 4°C.

[0010] Furthermore, a SSC-H / FL1-H scatter plot was established in the analysis software, and the forward angle scattered light FL1-H and side angle scattered light SSC-H of the flow cytometer were adjusted and determined.

[0011] Furthermore, all consumables and supplies used in this experiment need to be strictly sterilized.

[0012] Furthermore, the isolated high-concentration virus particles can be stably stored in SM buffer at 4°C for more than one month.

[0013] Compared with the prior art, the instruments and equipment used in the present invention are easier to obtain. Compared with the method of separating virus particles by ultracentrifugation, this method does not require an ultracentrifuge, and the reagent preparation method is simple, which greatly reduces the difficulty of reproducing the experimental method. The yield of virus particles separated by ultracentrifugation is equivalent to that by ultracentrifugation. It provides a good basis for studying the composition of the virus group in pig intestinal microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the process of enriching bacteriophage particles in pig feces samples according to the present invention.

[0015] Figure 2 It is a diagram of the precipitation state of the phage particles enriched in pig feces of the present invention.

[0016] Figure 3 It is a state diagram of the suspension of phage particles enriched in pig feces of the present invention.

[0017] Figure 4 It is the result of flow sorting the bacteriophage particles in the pig feces sample prepared by the present invention using a flow analyzer. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the embodiments and drawings, but is not intended to limit the protection scope of the present invention.

[0019] Experimental instruments and reagents: clean bench; pipette (Gilson); sterile pipette tip; 5 ml centrifuge tube (BD Falcon); desktop centrifuge (Sorvall Legend RT, Kendro); 2 ml grinding tube (BD Falcon); grinding zirconium beads; SM buffer; PEG solution; 0.42 μm and 0.22 μm (MF-Millipore™) microporous filter membrane; 10 ml sterile syringe; SYBR™ Gold nucleic acid gel stain (10,000X concentrate); chloroform (CHCl3). Example

[0020] 1. Enrichment of phage particles from pig feces samples (1.1) If Figure 1As shown, sterile zirconium beads and 1 mL of SM buffer were added to a centrifuge tube containing 1.0 g of feces, followed by vortexing for 5 min using a high-speed grinder and then transferred to a 15 mL centrifuge tube. SM buffer was then added to 10 mL; (1.2) Use a high-speed refrigerated centrifuge to centrifuge the sample at 6600 rpm and 4°C for 30 min. Perform a preliminary centrifugation to remove most of the fecal sample sediment.

[0021] (1.3) After centrifugation, take the supernatant and transfer it to a new centrifuge tube. Continue centrifugation in a high-speed refrigerated centrifuge (16,000 × g, 4 °C, 10 min). (1.4) If Figure 1 As shown, step (3) was repeated 4 times, and centrifugation was repeated to remove most of the sediment in the stool sample; (1.5) After completion, transfer the supernatant to a new sterile 50 ml centrifuge tube, filter through 0.45 μm and 0.22 μm microporous filter membranes in sequence, and collect the filtrate into a new centrifuge tube; (1.6) Add 5X PEG8000 NaCl to the filtrate so that the volume ratio of 5X PEG8000 NaCl to the filtrate is 1:4; (1.7) After the sample was allowed to stand at 4°C for 1 hour, it was centrifuged at 3400 g for 60 min at 4°C using a high-speed refrigerated centrifuge. Figure 2 As shown, the viral particle pellet was isolated.

[0022] (1.8) After centrifugation, discard the supernatant and resuspend the precipitate with an appropriate amount of SM buffer; (1.9) Repeat steps (1.6), (1.7) and (1.8); (1.10) Add CHCl3 to the filtrate so that the volume ratio of CHCl3 to filtrate is 1:4; incubate at room temperature (20℃~25℃) for 10 min to remove remaining bacteria and porcine intestinal cell fragments.

[0023] (1.11) Use a high-speed refrigerated centrifuge to centrifuge the sample at 5000g, 4°C for 15 min. The aqueous phase obtained by centrifugation is the high-concentration virus particle concentrate. Transfer the aqueous phase obtained by centrifugation to a new centrifuge tube. Figure 3 As shown, a high concentration of virus particle suspension was isolated.

[0024] 2. Counting of phages after enrichment (2.1) If Figure 1 SYBR™ Gold Nucleic Acid Gel Stain (10,000X concentrate) was diluted to 100X in dimethyl sulfoxide (DMSO) as shown.

[0025] (2.2) Repeat steps (1.1) to (1.9) and add SYBR™ Gold dye to the concentrated solution so that the ratio of the diluted dye volume to the concentrated solution volume is 1:99; (2.3) Incubate at 37°C in the dark for 1 h; (2.4) Add 5X PEG8000 NaCl to make the volume ratio of 5X PEG8000 NaCl to filtrate 1:4. Centrifuge at 23000g for 20 min at 4°C. Discard the supernatant and resuspend in SM buffer; (2.5) Repeat the above (2.4) to wash away excess dye to avoid false positive signals (2.6) If Figure 1 As shown, the stained virus particle concentrate was diluted 10, 100 and 1000 times with SM buffer. When the flow cytometer was used for detection, the side angle SSC-H and channel FL1-H were selected for detection; before the measurement, the flow cytometer automatically cleaned the inside to make the pipeline unobstructed and the nozzle clean; the stained sample was loaded, and the SSC-H / FL1-H scatter plot was established in the analysis software, and the forward angle scattered light FL1-H and side angle scattered light SSC-H of the flow cytometer were adjusted and determined. The sorting results of the positive virus particles by flow cytometry are shown in FIG. Figure 4 The number of positive phage particles at different gradient concentrations was counted, and the number of phages isolated from each gram of pig feces was converted and shown in Table 1.

[0026] Example

[0027] The difference from Example 1 is that the number of centrifugation times of the stool sample supernatant is increased, and there is no difference in other steps. There is no difference in the number of positive phage particles finally separated. Example

[0028] The difference from Example 1 is that the incubation condition of SYBR™ Gold dye on phage particles was changed to overnight at 4°C, and there was no difference in other steps. There was no difference in the number of positive phage particles finally isolated. Example

[0029] The difference from Example 1 is that the stool sample supernatant obtained after centrifugation can be directly filtered with a 0.22 μm microporous membrane, without the need for 0.45 μm microporous membrane filtration. There is no difference in the number of positive phage particles finally separated. Example

[0030] The difference from Example 1 is that the feces sample used is mouse feces. The number of positive phage particles finally separated is quite different from that of pig feces, indicating that different types of feces samples contain different numbers of phages.

[0031] The embodiments described above can be further combined or replaced, and the embodiments are only descriptions of preferred embodiments of the present invention, and do not limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention belong to the protection scope of the present invention. The protection scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for efficiently enriching bacteriophage particles in pig feces samples, characterized in that: The pig feces sample was fully dissolved in SM buffer, i.e., 100 mM NaCl, 8 mM MgSO4, 50 mM Tris, pH 7.5, and initially centrifuged to collect the supernatant; the supernatant was filtered with a 0.22 μm microporous filter membrane; the filtrate was collected and PEG 8000 and 0.6% NaCl with a final concentration of 20% by mass were added; it was allowed to stand at 4°C for more than 1 hour; then it was centrifuged at 4°C to precipitate PEG and virus particles; the supernatant was discarded and the precipitated material was resuspended with SM buffer; the above centrifugation, PEG, NaCl treatment and SM buffer resuspension steps were repeated; chloroform was added with a final concentration of 20%, and incubated at 20°C-25°C for 10 min; then the aqueous phase was separated by centrifugation, and the aqueous phase was transferred to a 1.5 mL centrifuge tube. The resulting aqueous phase was a high-concentration virus particle concentrate.

2. The method according to claim 1, characterized in that the steps include: (1.1) First, add sterile zirconium beads and 1 mL of SM buffer to a centrifuge tube containing 1.0 g of feces, then vortex for 5 min using a high-speed grinder, and then transfer to a 15 mL centrifuge tube; then add SM buffer to 10 mL; (1.2) Centrifuge the sample at 6600 rpm and 4 °C for 30 min using a high-speed refrigerated centrifuge; (1.3) After centrifugation, take the supernatant and transfer it to a new centrifuge tube. Centrifuge it in a high-speed refrigerated centrifuge at 16,000 × g, 4°C, for 10 min. (1.4) Repeat step (1.3) 3 to 4 times until there is no precipitation in the sample; (1.5) After completion, transfer the supernatant to a new sterile 50 ml centrifuge tube, filter through 0.45 μm and 0.22 μm microporous membranes in sequence, and collect the filtrate into a new centrifuge tube; (1.6) Add 5X PEG8000 NaCl to the filtrate so that the volume ratio of 5X PEG8000 NaCl to the filtrate is 1:4; (1.7) After the sample was allowed to stand at 4°C for 1 hour, it was centrifuged at 3400 g and 4°C for 60 minutes using a high-speed refrigerated centrifuge; (1.8) After centrifugation, discard the supernatant and resuspend the precipitate with an appropriate amount of SM buffer; (1.9) Repeat steps (1.6), (1.7) and (1.8); (1.10) Add CHCl3 to the filtrate to make the volume ratio of CHCl3 to filtrate 1:4; incubate at 20℃~25℃ for 10min to remove the remaining bacteria, porcine intestinal cell debris and PEG. Transfer the aqueous phase obtained by centrifugation to a new 1.5mL centrifuge tube; the aqueous phase is the high-concentration phage particle concentrate; (1.11) Use a high-speed refrigerated centrifuge to centrifuge the sample at 5000g, 4°C for 15 min; the aqueous phase obtained by centrifugation is the high-concentration virus particle concentrate.

3. The method according to claim 1, characterized in that After enrichment, the phages are counted. The steps include: (2.1) Dilute SYBR™ Gold Nucleic Acid Gel Stain, 10,000X concentrate, to 100X using dimethyl sulfoxide. (2.2) Add SYBR™ Gold dye to the virus particle concentrate so that the ratio of the diluted dye volume to the concentrate volume is 1:99; (2.3) Incubate at 37°C in the dark for 1 hour or at 4°C overnight; (2.4) Add 5X PEG 8000 NaCl to make the volume ratio of 5X PEG8000 NaCl to filtrate 1:4; centrifuge at 23000g for 20 min at 4°C; discard the supernatant and resuspend in SM buffer; (2.5) Add 5X PEG8000 NaCl again and centrifuge at 23000g for 20 min. Discard the supernatant and resuspend in SM buffer to wash away excess dye and avoid false positive signals. (2.6) The stained virus particle concentrate was diluted 10, 100 and 1000 times with SM buffer. When the flow cytometer was used for detection, the side angle SSC-H and channel FL1-H were selected for detection. The stained samples were loaded, and a SSC-H / FL1-H scatter plot was established in the analysis software. The forward angle scatter light FL1-H and side angle scatter light SSC-H of the flow cytometer were adjusted and determined. The number of positive phage particles at different gradient concentrations was counted and converted into the number of phages isolated from each gram of pig feces.

4. The method according to claim 1, characterized in that: The preparation method of SM buffer is as follows: 2.0 g of MgSO4·7H2O, 0.1 g of gelatin, 5.8 g of NaCl and 50 mL of 1M Tris-HCl pH=7.5 are mixed, and the volume is made up to 1000 mL with double distilled water; after high temperature and high pressure sterilization, cooling is the SM buffer, and it is stored at 4°C.

5. The method according to claim 1, characterized in that The preparation method of 5X PEG8000 NaCl is as follows: PEG8000 250g and NaCl 43.83g are mixed, and the volume is adjusted to 1000mL with double distilled water; after high temperature and high pressure sterilization, cooling is 5XPEG8000 NaCl solution, and it is stored at 4°C.

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