A method for isolating solanacearum phage and its application

Abstract

The present invention relates to the field of biological detection technology, and in particular to a method for isolating a solanacearum phage and its application. The method is based on the co-culture of host bacteria and phage, and the addition of a resin azure indicator to detect the turbidity OD of the bacteria. 600 The advantages are: 1. Adding resin azure can cause redox reaction and color change during bacterial growth, making the detection of bacterial activity more sensitive and accurate for bacterial solution OD 600 The detection is also more sensitive; 2. Liquid culture medium can be used to separate Ralstonia solanacearum phages by monitoring bacterial turbidity OD 600 The bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance can be determined from two aspects: the bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance, thereby screening out high-quality phage combinations that are highly effective in killing bacteria and not prone to developing resistance; 3. This method can also be used for the screening of highly effective bactericidal phage groups and for antibacterial experiments involving the combination of phages with other drugs such as antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for isolating a solanacearum phage and an application thereof. Background Art

[0002] Ralstonia solanacearum (hereinafter referred to as Ralstonia solanacearum) causes serious bacterial wilt disease worldwide, and statistics show that it causes economic losses of approximately US$12 billion each year, ranking second in the world (Mansfield et al.). Due to the huge economic losses it causes, bacterial wilt has become an important target disease for phage therapy.

[0003] Phages used to control bacterial wilt are all virulent phages, which can quickly lyse Ralstonia solanacearum in a shorter time, thus achieving the purpose of rapidly reducing its population. There have been many reports on the use of phage therapy to target and inactivate Ralstonia solanacearum in soil-plant systems. Wang et al. found that the application of lytic phages of Ralstonia solanacearum into the soil can effectively reduce the population of Ralstonia solanacearum in the tomato rhizosphere, and the prevention effect on tomato bacterial wilt reached 83.4%. Askora et al. isolated and purified four Ralstonia solanacearum phages (φRSL, φRSA, φRSM and φRSS) from farmland soil. The phage stock solution was treated by root irrigation or injected into the main stem of the plant 1 cm from the soil surface, which could significantly reduce the incidence and disease index of tomato and tobacco bacterial wilt. Tanaka et al. reported the control effect of tobacco bacterial wilt using the non-toxic R. solanacearum M4S strain and the phage infected with the strain. They found that the wilt incidence of the blank control reached 95.8%, the incidence of M4S alone was 39.5%, and the incidence of M4S and phage at the same time was only 14.5%, showing a good control effect. Fujiwara et al. used three virulent phages φRSL1, φRSA1 and φRSB1 to treat Ralstonia solanacearum. The results showed that φRSA1 and φRSB1 alone or in combination with other phages could quickly reduce the population density of Ralstonia solanacearum, and all treated tomato seedlings did not show wilting symptoms during the experiment, while the tomato seedlings not treated with φRSL1 wilted on the 18th day. Bhunchoth et al. also found that the short-tailed phage φJ2 and the muscle-tailed phage φRSF1 can effectively reduce the number of Ralstonia solanacearum in the soil, thereby playing a role in preventing and controlling tomato bacterial wilt. Gao Miao et al. isolated a bacteriophage φRS-1 that can lyse Ralstonia solanacearum from tobacco field soil. The phage has the characteristics of a short incubation period, strong lysis ability and long duration of lysis activity, and has the potential to be developed into a phage preparation against Ralstonia solanacearum.

[0004] The reduction of the number of pathogenic bacteria by phages is the basis for phage therapy to work, which is of great significance to the pathogenic system that is dependent on the number of pathogenic bacteria. The expression of pathogenic characteristics of some pathogenic bacteria depends on the population size, that is, only when the population size reaches a certain threshold can the pathogenic system be activated, virulence factors be secreted and symptoms be caused. Therefore, phages quickly infect and lyse host cells, which not only directly reduces the possibility of pathogenic bacteria invading the host, but also indirectly reduces its pathogenicity, thereby effectively controlling the occurrence of bacterial wilt and reducing losses due to disease. However, the key to effective phage treatment is to be able to screen and match phages that can lyse the bacteria or have a wide host spectrum. At present, the commonly used phage separation and matching method for bacterial wilt phages is the double-layer agar method (DLA), that is, different phages are spotted on the surface of the target bacterial plate, and the lysis power of the phage is judged by observing whether plaques are formed and the transparency of the plaques. Since most of the Ralstonia solanacearum phages are isolated from the soil, the phage content in the soil is low and the bacterial flora is complex, resulting in a low Ralstonia solanacearum phage isolation rate and a high contamination rate. In addition, the culture time of Ralstonia solanacearum is relatively long, averaging 24 to 48 hours. Therefore, the conventional double-layer agar method is not easy to achieve high-throughput phage screening, and the observation time is relatively long, which increases the time cost and labor cost. Summary of the invention

[0005] In view of this, the present invention provides a method for isolating Ralstonia solanacearum phage and its application, so as to solve the problems of low separation rate, small screening throughput, difficulty in evaluating the phage lysis effect and phage tolerance in the Ralstonia solanacearum phage process, and achieve the purpose of efficient and high-throughput screening of Ralstonia solanacearum phage.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a screening method for Ralstonia solanacearum phage or a combination of Ralstonia solanacearum phages, comprising: mixing a sample to be separated with Ralstonia solanacearum and a resin azure solution, co-culturing, and obtaining an OD 600 , according to OD 600 The Ralstonia solanacearum phage or Ralstonia solanacearum phage is screened and obtained.

[0008] In some specific embodiments of the present invention, the above screening method comprises the following steps:

[0009] S1. Mix the sample with the buffer solution, shake it, take the supernatant, filter and sterilize it to obtain the extract;

[0010] S2, mixing Ralstonia solanacearum with the extract, co-culturing, filtering, and obtaining enrichment solution 1;

[0011] S3, mixing Ralstonia solanacearum with the enrichment solution 1, co-culturing, filtering, and obtaining enrichment solution 2;

[0012] S4, mixing Ralstonia solanacearum with the enrichment solution 2 and the resin azure solution, co-culturing, and obtaining OD 600 , according to OD 600 The Ralstonia solanacearum phage or Ralstonia solanacearum phage is screened and obtained.

[0013] In some specific embodiments of the present invention, the co-cultivation conditions in S2 and S3 of the above screening method are 28°C, 29°C, 30°C, 31°C or 32°C, 200 rpm, 210 rpm, 220 rpm, 230 rpm or 240 rpm.

[0014] In some specific embodiments of the present invention, the co-cultivation conditions in S4 of the above screening method are 28°C, 29°C, 30°C, 31°C or 32°C, 450 rpm, 460 rpm, 470 rpm, 480 rpm, 490 rpm, 500 rpm, 510rpm, 520 rpm, 530 rpm, 540 rpm or 550 rpm.

[0015] In some specific embodiments of the present invention, the co-cultivation time in S4 of the above screening method is 5 h.

[0016] In some specific embodiments of the present invention, the filtration in the above screening method is filtered with a pore size of 0.22 μm.

[0017] In some specific embodiments of the present invention, the concentration of the resin azure solution in the above screening method is 0.6% (0.6 g / 100 mL).

[0018] In some specific embodiments of the present invention, S4 of the above screening method is:

[0019] Ralstonia solanacearum was mixed with the enrichment solution 2 and the resin azure solution, and co-cultured to form an experimental group;

[0020] Ralstonia solanacearum was mixed with resin azure solution and co-cultured as a control group;

[0021] Obtain the OD of the experimental group and the control group respectively. 600 If the experimental group OD 600 Compared with the control group OD 600 If the decrease is ≥40%, the samples containing virulent phages are screened out.

[0022] The present invention also provides a method for obtaining the tolerance time of Ralstonia solanacearum to bacteriophage, comprising obtaining the OD 600 , when OD 600 The time it takes to reach 1.5 for the first time is the duration of tolerance of Ralstonia solanacearum to the bacteriophage.

[0023] The present invention also provides a method for isolating Ralstonia solanacearum bacteriophage, comprising screening a sample containing virulent bacteriophage based on the above screening method, and then isolating the Ralstonia solanacearum bacteriophage based on a double-layer agar method.

[0024] The method for isolating the solanacearum phage of the present invention and its application have the following effects:

[0025] 1. Adding resin Azure can cause redox reaction and color change during bacterial growth, making the detection of bacterial activity more sensitive and accurate for bacterial solution OD 600 The detection is also more sensitive; 2. Liquid culture medium can be used to separate Ralstonia solanacearum phages by monitoring bacterial turbidity OD 600 The bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance can be determined from two aspects: the bactericidal efficiency of phages and the time it takes for bacteria to develop phage resistance, thereby screening out high-quality phage combinations that are highly effective in killing bacteria and not prone to developing resistance; 3. This method can also be used for the screening of highly effective bactericidal phage groups and for antibacterial experiments involving the combination of phages with other drugs such as antibiotics.

[0026] In the present invention, the phage separation sample is treated twice during the culture process to increase the concentration of the phage after culture, and then the phage and bacteria are co-cultured in a 96-well plate and resin azure is used as an indicator to detect the bacterial turbidity OD during the culture process. 600 The addition of the resin azure indicator can improve the detection sensitivity of bacterial turbidity changes, and then judge the bacteriophage bacteriocidal effect on Ralstonia solanacearum and the bacterial tolerance to Ralstonia solanacearum phage, thereby screening out the phage that has the most obvious inhibition on bacterial growth and is not easy to produce tolerance to bacteria. The present invention can also be applied to the detection of antibiotics, antimicrobial peptides, lytic enzymes and other phage interactions, greatly shortening the cultivation time of Ralstonia solanacearum, and more conveniently and quickly realizing the screening of batches of virulent Ralstonia solanacearum phages. It is of great significance to quickly respond to the rapid phage matching of Ralstonia solanacearum in the field and the matching of cocktails. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 The phage screening process is shown in FIG.

[0029] Figure 2Shows a single colony of Ralstonia solanacearum on TTC medium;

[0030] Figure 3 Example 1 Turbidity OD after Ralstonia solanacearum phage infected bacteria 600 Trends over time;

[0031] Figure 4 Example 1 shows plaques using a double-layer agar plate method;

[0032] Figure 5 Schematic diagram showing the time point determination for screening solanacearum phage by liquid method in Example 1;

[0033] Figure 6 The growth curves of the combination of bacteriophages and the interaction of a single bacteriophage with bacteria in Example 2 are shown;

[0034] Figure 7 The bacterial growth curve after the phage and antibiotics act together in Example 3 is shown. DETAILED DESCRIPTION

[0035] The present invention discloses a method for isolating a solanacearum phage and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0036] The present invention discloses a method for isolating a solanacearum phage and its application, wherein a resin azurin indicator is added during the separation process, which can not only improve the isolation rate of the solanacearum phage but also achieve the purpose of high-throughput screening of the solanacearum phage, and can also be used to screen the solanacearum phage according to the culture time, bacterial OD 600 By measuring the changes in the values, we can screen for phages with high bactericidal efficiency and high-quality phages or phage combinations that are not prone to tolerance.

[0037] The resin azure is a water-soluble dye that is non-toxic to cells. After entering the cells, it undergoes an oxidation-reduction reaction under the action of mitochondrial enzymes, thereby producing a color change of "blue-purple-pink-colorless". Among them, blue represents the oxidation state of the resin azure, which can be further reduced to pink resorcinol and colorless hydroresorcinol, and the color change of the resin azure can reflect the strength of cell vitality.

[0038] Theoretically, there is an interaction between phages and bacteria during the separation process of phages, which causes the bacterial content in the phage sample to decrease, that is, the cell viability is reduced. Therefore, the present invention proposes that by adding resin azure, it is helpful to more quickly determine the existence of phages during the phage separation process and more quickly screen out high-quality phages that are not easy to cause tolerance to Ralstonia solanacearum. The specific technical solution is as follows:

[0039] The method for separating the solanacearum phage comprises the following steps:

[0040] S1. Soil sample processing: soaking in SM solution, centrifugation, filtration and sterilization;

[0041] S2, the first co-culture of host bacteria and phage;

[0042] S3, the second co-culture of host bacteria and phage;

[0043] S4. The phage was co-cultured with a single host bacterium in a 96-well microplate, and the resin azure indicator was added to detect the bacterial turbidity OD 600 .

[0044] According to some embodiments of the present invention, the SM solution immersion ratio in step S1 is sample:SM solution = 1:10, and the leaching is shaken at 170 rpm for 2 h.

[0045] According to some embodiments of the present invention, the centrifugation in S1 is 11000 rpm for 15 min.

[0046] According to some embodiments of the present invention, the culture temperature in S1-S4 is 30°C.

[0047] According to some embodiments of the present invention, the culturing time in S2 and S3 is ≥ 12 h.

[0048] According to some embodiments of the present invention, the culture medium in S2 and S3 is 2×NB.

[0049] According to some embodiments of the present invention, 2 μL of 0.6% resin azure solution is added to the culture medium in S4.

[0050] According to some embodiments of the present invention, the co-cultivation process in S4 requires shaking and standing in a single cycle.

[0051] According to some embodiments of the present invention, the oscillation time in a single cycle described in S4 is 30 min.

[0052] According to some embodiments of the present invention, the standing time in a single cycle described in S2 is 2 to 3 min.

[0053] According to some embodiments of the present invention, the oscillation speed in a single cycle described in S2 is 500 rpm

[0054] According to some embodiments of the present invention, the oscillation rate during the leaching process is 220 rpm to 300 rpm.

[0055] According to some embodiments of the present invention, the culturing time in S4 is 4 to 5 h.

[0056] According to some embodiments of the present invention, the sterilization is performed by filtering with a 0.22 μm filter membrane.

[0057] According to some embodiments of the present invention, the lysis ability of the bacteriophage to Ralstonia solanacearum is defined as:

[0058] (1) [((Control group OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%]≥80% is strong cleavage, recorded as "+++";

[0059] (2) 40%≤[((control group OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%] < 80% is moderate cleavage, recorded as "++";

[0060] (3) 20%≤[((control group OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%] < 40% was mild lysis, recorded as “+”;

[0061] (4) [((Control group OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%]<20% means no cleavage and is recorded as “-”.

[0062] According to some embodiments of the present invention, the strong lysis ability of the bacteriophage to Ralstonia solanacearum is defined as the turbidity OD of the bacteria in the experimental group. 600 Compared with the control group, the OD 600 Compared with the previous study, the difference was ≥40%.

[0063] According to some embodiments of the present invention, the phage lysis ability to Ralstonia solanacearum is defined as the detection OD 600 A control group needs to be set during the process to calibrate the background value.

[0064] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0065] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0066] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0067] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0068] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0069] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0070] The present invention will be further described below in conjunction with the embodiments.

[0071] Example 1

[0072] This embodiment is a method for isolating a solanacearum phage and its application, which consists of the following steps:

[0073] S1. Soil sample processing

[0074] Soil or sewage samples were mixed with SM buffer at a ratio of 1:10 and shaken for 2 h to fully extract the bacteriophages in the soil. 5 mL of the supernatant was taken from the suspension and centrifuged at 11,000 rpm for 15 min. The suspension was sterilized by filtration using a 0.22 μL filter membrane to obtain a sterile extract.

[0075] S2. First co-culture of host bacteria and phage

[0076] Take the preserved strain to be tested and inoculate it in three zones on the TTC medium plate, incubate it at 28-30℃ for 48 h, and obtain a single colony ( Figure 2 ), pick a single colony and inoculate it into 5 mL NB broth medium, and culture it at 28-30℃ constant temperature shaker at 220 rpm / min until the logarithmic phase to obtain bacterial liquid.

[0077] Take 2 mL of the extract from S1, 200 μL of the above bacterial solution and 5 mL of 2×NB broth culture medium in a 10 mL centrifuge tube, and culture at 30°C, 220 rpm and shaking overnight to obtain the enrichment solution; centrifuge the enrichment solution at 11000 rpm for 15 min and filter it with a 0.22 μm filter for sterilization.

[0078] S3. Second co-culture of host bacteria and phage

[0079] Take 2 mL of S2 sterile enrichment solution, 200 μL of bacterial solution and 5 mL of 2× broth culture medium in a 10 mL centrifuge tube, and culture at 30°C, 220 rpm and shaking overnight to obtain the second enrichment solution. Centrifuge the second enrichment solution at 11,000 rpm for 15 min, filter and sterilize it with a 0.22 μm filter, transfer the filtrate to a new sterile centrifuge tube, and store it at 2-8°C for later use.

[0080] S4. Co-culture the host bacteria with the enrichment solution in a 96-well microplate and detect the bacterial turbidity OD 600

[0081] Preparation of 0.6% resin azure solution: Take 0.667 g of 90% resin azure (MAKELIN ® ) solution was added into 100 mL of NB liquid culture medium of Ralstonia solanacearum and mixed thoroughly to prepare a 0.6% (6 g / L) resin azure solution.

[0082] Use a dispenser to take 5 μL / well of the phage enrichment solution prepared in S3 and add it to a 96-well plate. Add 2 μL / well of resin azure solution and add equal amounts of different host bacterial solutions (about 10 6 CFU / well), with a total volume of 200 μL. A control group without phage was set up, and after the operation, the 96-well plate was placed in a microbial growth curve analyzer for culture. At the same time, a group of phage mixtures and host bacteria with known host bacteria were selected to set up a resin azure group, a methylene blue group, and a control group without dye, and phages were isolated using the 96-microwell culture method to observe the sensitivity of phage isolation and the difference in separation quantity between different dyes.

[0083] The microbial growth curve analyzer was set up under the following conditions: 30°C, pre-shaking at 1000 rpm, shaking time of 10 s; shaker speed of 500 rpm / min, sampling interval of 30 min, sampling delay of 2 s; reading bacterial turbidity OD 600 , continuous monitoring for 7 h.

[0084] S5. Phage purification

[0085] Take the turbidity OD of the co-cultured bacteria 600 Compared with the control group, the OD 600 Dilute the phage incubation solution with a 10-fold ratio to a suitable gradient, take 120 μL of phage extract and 120 μL of bacterial solution and mix them evenly, incubate at 30°C for 5 min, add them to the upper culture medium at about 50°C, mix them evenly and quickly pour them into the plate of the lower culture medium, shake them evenly, and invert them in a 30°C incubator after solidification to obtain a double-layer plate with transparent plaques. Use sterile tweezers to pick out a single plaque together with the upper culture medium attached to it, put them into a centrifuge tube containing 1 mL of NB broth, and crush the attached culture medium with tweezers. Place the centrifuge tube on a shaker at 28°C and 220 rpm for at least 30 min for standby use. Repeat the above steps and purify at least 2 times until the plaques are consistent in morphology, size, and transparency ( Figure 4 ).

[0086] S6. Data Analysis

[0087] According to the absorbance monitored by the microbial growth curve analyzer, the absorbance OD 600 The numerical values ​​are calculated and matched with the phage lysis ability. The growth curve of the bacterial solution in each well is drawn at the same time. The growth curve of the co-culture group with added phage is superimposed and compared with the growth curve of the control group to analyze whether the phage added to each well can effectively lyse the host bacteria.

[0088] In step S4, the conventional double-layer plate method was simultaneously used to separate Ralstonia solanacearum phages to compare the separation efficiency and accuracy of the phages.

[0089] Results: The OD of Ralstonia solanacearum was 600 The time when the value reaches 1.5 is the time when the bacteria tolerate the bacteriophage. 600 - Experimental group OD 600 ) / control group OD 600 ×100%, and determine whether there is a phage in the phage sample library that can lyse the bacteria and the tolerance of the phage to the strain based on the percentage range. According to the data in Tables 1 and 2, according to the OD 600Reducing the ratio can determine whether the phage is isolated and the lysis performance of different phages can be clearly observed. The lysis ability of the phage is stable during the culture period of 2.5 h to 5 h. When the culture time is ≥6 h, some Ralstonia solanacearum that is resistant to phage begins to be produced, which affects the actual observation results. Therefore, in the method for isolating Ralstonia solanacearum phage of the present invention and its application, the culture time is determined to be 5 h in the process of screening phages. According to the data in Table 3, compared with the conventional method, the 96-microwell co-culture method can shorten the culture time of phage isolation by 1 to 1.5 h, and the number of high-titer phages isolated by the 96-microwell culture method is more than that of the conventional method, and the phage purification and preservation rate is 30% higher than that of the conventional method. According to Figure 5 The curve of the interaction between the phage sample library and the host bacteria shows that the same strain of Ralstonia solanacearum has different tolerance times to different phages. 600 When the value is 1.5, the tolerance time of Ralstonia solanacearum to the bacteriophage is mainly distributed in 3.5 h, 7.5~10 h, and a very small amount in 17 h and later. 600 The length of the culture time when the value reaches 1.5 is used to preliminarily determine the tolerance of Ralstonia solanacearum to phages, which is conducive to the separation of phages with better bacterial tolerance. As shown in Table 4, the addition of resin azure to the 96-micropore phage separation and culture method can significantly improve the separation sensitivity of phages. Therefore, the method for separating Ralstonia solanacearum phages and its application in the present invention can improve the separation efficiency and quality of Ralstonia solanacearum phages.

[0090] Table 1: OD at the same culture time 600 Reduction percentage

[0091]

[0092] The calculation formula is: (control group OD 600 - Experimental group OD 600 ) / control group OD 600 ×100%

[0093] Table 2: Lysis ability of different phages at the same culture time

[0094]

[0095] Table 3: Comparison of results between 96-well co-culture method and conventional separation method

[0096]

[0097] Table 4: Comparison of the effects of different stains on phage separation

[0098]

[0099] Example 2

[0100] This embodiment is a method for efficiently and high-throughput screening of Ralstonia solanacearum phages, which consists of the following steps:

[0101] S1. Preparation of Ralstonia solanacearum phage

[0102] 1. Bacterial recovery and bacterial solution preparation

[0103] Take the preserved strain to be tested and inoculate it in three zones on the TTC medium plate, place it at 28-30℃ for 48 hours, obtain a single colony, pick a single colony and inoculate it in 5 mL NB broth medium, and culture it in a constant temperature shaker at 28-30℃ at 220 rpm / min until the logarithmic phase. Take 5 μL of the logarithmic phase bacterial solution and add it to a 96-well plate containing 190 μL of NB broth medium for later use.

[0104] 2. Preparation of Ralstonia solanacearum phage

[0105] Take 100 μL of frozen phage solution to be tested and 100 μL of host bacterial solution in 5 mL NB, set up a bacterial solution control group, and culture in a constant temperature incubator at 28-30℃ at 220 rpm / min. Observe that the amplified solution changes from clear to turbid to clear again, and the amplified solution is clearer than the control bacterial solution. Centrifuge the amplified solution at 11000 rpm for 5 min, and then filter it with a 0.22 μm filter to obtain the phage filtrate (titer>10 8 PFU / mL).

[0106] S2. High-throughput screening of Ralstonia solanacearum phage

[0107] Prepare the resin azure solution according to S4 in Example 1. Use a pipette to pipette 5 μL / well of the prepared phage library into the 96-well plate containing the bacterial solution prepared in step S1 of this example. The ratio of different phages in the phage combination is 1:1. Add 2 μL / well of resin azure, and the total volume is 200 μL. Set up a single phage control group and a bacterial solution control group (such as Figure 6 After the operation, the 96-well plate was placed in a microbial growth curve analyzer for culture.

[0108] The microbial growth curve analyzer was set up under the following conditions: 30°C, pre-shaking at 1000 rpm, shaking time of 10 s; shaker speed of 500 rpm / min, sampling interval of 30 min, sampling delay of 2 s; reading bacterial turbidity OD 600 , continuous monitoring.

[0109] Bacterial OD 600 The time point from 1.0 to 1.5 is the standard for determining the tolerance of bacteria to bacteriophages, and this time point is the tolerance time of bacteria to bacteriophages.

[0110] S3. Data Analysis

[0111] According to the absorbance monitored by the microbial growth curve analyzer, the growth curve of each well of bacterial solution was drawn, and the growth curve of the experimental group with the addition of phage combination was superimposed and compared with the growth curve of the control group with the addition of single phage. It was analyzed whether the addition of phage combination to each well had a better lysis effect than the addition of single phage and the time that bacteria were tolerated. The above information was counted and recorded.

[0112] Results: Figure 6 As shown, the OD of the bacterial solution control group 600 When the OD reached 1.5, it took about 2.5 h. When single phage phageA and phageB were added, the OD 600 When the OD value reached 1.5, it took about 9.2 h and 12.5 h, respectively, indicating that the addition of a single phage had a certain inhibitory effect on bacteria, but the bacteria had a short tolerance to the phage. The addition of the phage combination phageA+phageB showed that the growth of bacteria was continuously inhibited within 25 h of culture, and the OD value was 1.5. 600 The time to reach 1.5 is greater than 25 h, indicating that the phage combination has a better antibacterial effect and is less likely to be tolerated by bacteria.

[0113] Example 3

[0114] This embodiment is a method for efficiently and high-throughput screening of Ralstonia solanacearum phages, which consists of the following steps:

[0115] S1. Preparation of Ralstonia solanacearum phage library

[0116] 1. Bacterial recovery and bacterial solution preparation

[0117] Take the preserved strain to be tested and inoculate it in three zones on the TTC medium plate, place it at 28-30℃ for 48 h, and obtain a single colony. Pick a single colony and inoculate it in 5 mL NB broth medium, and culture it at 28-30℃ constant temperature shaker at 220 rpm / min until the logarithmic phase (OD 600 5 μL of the logarithmic phase bacterial solution was added to a 96-well plate containing 190 μL of NB broth medium for later use.

[0118] 2. Preparation of Ralstonia solanacearum phage

[0119] Take 100 μL of frozen phage solution to be tested and 100 μL of host bacterial solution in 5 mL NB, set up a bacterial solution control group, and culture in a constant temperature incubator at 28-30℃ at 220 rpm / min. Observe that the amplified solution changes from clear to turbid to clear again, and the amplified solution is clearer than the control bacterial solution. Centrifuge the amplified solution at 11000 rpm for 5 min, and then filter it with a 0.22 μm filter to obtain the phage filtrate (titer>10 8 PFU / mL).

[0120] S2. High-throughput screening of Ralstonia solanacearum phage

[0121] Prepare the resin azure solution according to S4 in Example 1. Use a pipette to pipette 5 μL / well of the prepared phage library into the 96-well plate containing the bacterial solution prepared in step S1 of this example. The ratio of phage to antibiotic in the phage and antibiotic combination is 1:1 (volume ratio, dilute the antibiotic according to the usage and dosage, and mix it with the phage volume 1:1), add 2 μL / well of resin azure, and the total volume is 200 μL. Set a single phage as the control group and a bacterial solution control group. After the operation, the 96-well plate is placed in a microbial growth curve analyzer for culture.

[0122] The microbial growth curve analyzer was set up under the following conditions: 30°C, pre-shaking at 1000 rpm, shaking time of 10 s; shaker speed of 500 rpm / min, sampling interval of 30 min, sampling delay of 2 s; reading bacterial turbidity OD 600 , continuous monitoring.

[0123] Bacterial OD 600 The time point from 1.0 to 1.5 is the standard for determining the tolerance of bacteria to bacteriophages, and this time point is the tolerance time of bacteria to bacteriophages.

[0124] S3. Data Analysis

[0125] According to the absorbance monitored by the microbial growth curve analyzer, the growth curve of each well of the bacterial solution was drawn, and the growth curves of the antibiotics and phages, single phages, single antibiotics and the control group were superimposed and compared to analyze whether the combination of phages and antibiotics in each well had a better lysis effect than the addition of single phages or single antibiotics, and the time the bacteria were tolerated. The above information was counted and recorded.

[0126] Results: Figure 7 As shown, OD of bacterial control group, single phage group, and single antibiotic group 600 The time to reach 1.5 was about 4 h, 8.5 h, and 16.7 h, respectively, indicating that the single phage and single antibiotic group had a certain inhibitory effect on bacteria, and the single antibiotic group was better than the single phage group. The addition of phage and antibiotic combination continued to inhibit the growth of bacteria within 25 h of culture, and OD 600 The time to reach 1.5 is greater than 25 h. This indicates that the combination of bacteriophage and antibiotics has a better antibacterial effect than the use of bacteriophage or antibiotics alone and is less likely to be tolerated by bacteria.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for screening Ralstonia solanacearum phage, characterized in that: include: The sample to be separated was mixed with Ralstonia solanacearum and resin azure solution, co-cultured, and the OD 600 , according to OD 600 The Ralstonia solanacearum phage was obtained by screening.

2. The screening method according to claim 1, characterized in that The steps include: S1. Mix the sample with the buffer solution, shake it, take the supernatant, filter and sterilize it to obtain the extract; S2, mixing Ralstonia solanacearum with the extract, co-culturing, filtering, and obtaining enrichment solution 1; S3, mixing Ralstonia solanacearum with the enrichment solution 1, co-culturing, filtering, and obtaining enrichment solution 2; S4, mixing Ralstonia solanacearum with the enrichment solution 2 and the resin azure solution, co-culturing, and obtaining OD 600 , according to OD 600 The Ralstonia solanacearum phage was obtained by screening.

3. The screening method according to claim 2, characterized in that The conditions for the co-culture described in S2 and S3 were 28-32°C and 200-240 rpm.

4. The screening method according to claim 2 or 3, characterized in that The co-culture conditions described in S4 were 28-32°C and 450-550 rpm.

5. The screening method according to any one of claims 2 to 4, characterized in that The co-culture time described in S4 was 5 h.

6. The screening method according to any one of claims 2 to 5, characterized in that The filtration was performed with a pore size of 0.22 μm.

7. The screening method according to any one of claims 1 to 6, characterized in that The concentration of the resin azure solution is 0.6%.

8. The screening method according to any one of claims 1 to 7, characterized in that S4 is: Ralstonia solanacearum was mixed with the enrichment solution 2 and the resin azure solution, and co-cultured to form an experimental group; Ralstonia solanacearum was mixed with resin azure solution and co-cultured as a control group; Obtain the OD of the experimental group and the control group respectively. 600 If the OD of the experimental group 600 Compared with the control group, the OD 600 A decrease of ≥40% indicates that virulent phages exist in the sample and Ralstonia solanacearum phages are obtained.

9. A method for obtaining the tolerance duration of Ralstonia solanacearum to a bacteriophage or a combination of Ralstonia solanacearum phages, characterized in that: The OD is obtained based on the screening method according to any one of claims 1 to 7 600 , when OD 600 The time it takes to reach 1.5 hours for the first time is the tolerance time.

10. A method for isolating a Ralstonia solanacearum bacteriophage, characterized in that: A sample containing virulent phage is screened based on the screening method according to any one of claims 1 to 7, and then the Ralstonia solanacearum phage is separated based on the double-layer agar method.

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