CN114277084A - Method for identifying plague phage host spectrum based on micro liquid culture medium - Google Patents
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- 238000012360 testing method Methods 0.000 claims abstract description 38
- 241001515965 unidentified phage Species 0.000 claims abstract description 36
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- 239000007788 liquid Substances 0.000 claims abstract description 16
- 125000003831 tetrazolyl group Chemical group 0.000 claims abstract description 11
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Abstract
The invention discloses a method for identifying plague phage host spectra based on a trace liquid culture medium, which uses a high-throughput test that the liquid volume of the culture medium is small, each well is only 90ul, and 1 96-well plate can simultaneously monitor the interaction of a plurality of phages and tested bacteria. The mixed solution of bacteriophage and test bacteria added with tetrazolium-based dye in the micro-culture solution was used to automatically measure the growth of bacteria by color change of tetrazolium-based dye by means of the design of OmniLog TM system, i.e., relying on redox chemistry, using cellular respiration as a universal reporter. The OmniLog TM system has the advantages of being micro, rapid, sensitive, accurate and the like, compared with the traditional phage test, the OmniLog TM system has obvious advantages in real-time, continuous monitoring and automatic monitoring of phage infection and bacterial growth in the whole test process, and has good application value in research works such as plague phage separation, host spectrum identification, biological characteristic identification and the like.
Description
Technical Field
The invention relates to the technical field of pathogenic microorganisms, in particular to a method for identifying plague phage host spectra based on a trace liquid culture medium.
Background
Plague phages, as bacterial viruses of plague bacteria, are widely present in nature. It is a strictly parasitic bacterial virus, which is adsorbed on bacterial cells, must grow and propagate within the viable susceptible bacterial cells, and can lyse the host cells. There are many specific changes of phage in the infection, proliferation and maturation phases of phage-infected bacteria. The assay procedure for these changes is generally determined by liquid assays and semi-solid culture methods.
In which, the liquid assay generally monitors the decrease in optical density of the bacterial culture after infection, growth and lysis by the phage, so as to indirectly determine the lytic activity of the phage, the total volume of the culture fluid required for the liquid identification of the plague phage host spectrum is less than 5ml, but the optical density value may be increased by the bacterial cell debris generated by lysis, thereby masking or underestimating the true lytic activity of the phage. In addition, conventional overnight phage growth experiments give a false impression because turbidity in the culture broth after overnight incubation is mistaken for lysis, whereas in fact the phage has been completely lysed before bacterial growth has developed. Semi-solid culture methods include cross-strip assays, dot-drop methods, and plaque counting methods, which are capable of quantifying the lytic activity of a bacteriophage on its host bacterium. However, in current phage studies it was found that some wild-type plague phages were able to reduce the optical density of the culture broth containing the bacterial culture, but were unable to form plaques on semi-solid media, which does not necessarily mean that the phage was devoid of productive infection. The preparation process of the semisolid culture medium is complex, a bottom layer culture medium needs to be poured in a plate, a solidified upper layer culture medium needs to be melted in a later test, phage and detected bacteria need to be added into the plate at a proper temperature for a proper time, and the plate is moved to an incubator after being solidified. Only 1 phage was verified on the plate to correspond to 1 test bacterium.
In view of the above, both liquid and semi-solid culture assays are laborious, time-consuming and expensive, especially in high-throughput assays where multiple phage-bacteria interactions need to be monitored simultaneously.
Disclosure of Invention
Based on the technical problems, the invention aims to provide a method for identifying plague phage host spectra based on a micro liquid culture medium.
The invention provides a method for identifying plague phage host spectra based on a micro liquid culture medium, which comprises the following specific steps:
and 7, sealing the plate openings of the 96-hole micro-culture plates, placing the plates in an OmniLogTM system, operating at 33 ℃ for 48h for reading, and observing a kinetic graph of the phenotypic reaction of each group, thereby quantitatively determining the strength of the phenotypic reaction.
Further, in the step 5, the experimental plague bacteria or non-plague bacteria added in each row are sequentially plague vaccine strains EV76The bacillus subtilis comprises virulent plague bacillus 614F, yersinia pseudotuberculosis PTB3, yersinia pseudotuberculosis PTB5, escherichia coli V517 and enterocolitis bacteria 52302-2.
Further, in the step 6, the wild plague phages to be detected are 476 wild plague phages and 087 wild plague phages from plague areas in heaven county; wild type plague phage from 072204 plague provenance of the same country; the plague bacteriophage for laboratory diagnosis is from a professional laboratory of plague bacteria.
Compared with the prior art, the invention has the following beneficial effects:
the method for identifying the plague phage host spectrum based on the micro liquid culture medium has the advantages that the amount of the used culture medium liquid is small, each hole is only 90ul, and 1 96-hole plate can simultaneously monitor the high-throughput test of the interaction of a plurality of phages and tested bacteria. Adding tetrazole dyes into the culture solution, and automatically measuring the growth condition of the bacteria by the mixed solution of the phage and the tested bacteria through the design of an OmniLog TM system, namely, by relying on redox chemistry and adopting cell respiration as a universal reporter and through the color change of the tetrazole dyes. The OmniLog TM system has the advantages of being micro, rapid, sensitive, accurate and the like, compared with the traditional phage test, the OmniLog TM system has obvious advantages in real-time, continuous monitoring and automatic monitoring of phage infection and bacterial growth in the whole test process, and has good application value in research works such as plague phage separation, host spectrum identification, biological characteristic identification and the like.
Drawings
FIG. 1 shows the growth of 3 pestis phages on liquid medium (liquid method) after 18-20h at 28 ℃ and 37 ℃;
FIG. 2 shows the growth of 3 pestis phages on solid medium (drop method) after 24h at 28 ℃;
FIG. 3 shows the growth of 3 pestis phages on solid medium (drop method) after 24-48h at 37 ℃;
FIG. 4 is a 96-well microplate for the experiments of the invention;
FIG. 5 is a graph showing the growth of wild type pestivirus 476 after 48 hours at 33 ℃ and its color development;
FIG. 6 is a graph showing the growth of wild type plague phage 087 at 33 ℃ for 48 hours and its color development;
FIG. 7 is a graph showing the growth of a wild type plague phage 072204 after 48 hours at 33 ℃ and its color development;
FIG. 8 is a graph showing the growth of plague phage for laboratory diagnosis after 48 hours at 33 ℃ and its color development;
FIG. 9 shows that wild-type plague phage 476 formed plaques of uniform size after 3 purifications.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Optimization experiment of purification conditions of wild type plague bacteriophage
1. Purpose of experiment
The lysis conditions of 3 wild plague phages were tested by a drop method and a liquid method, respectively
2. Test object
The wild type plague phage was: pestis phage 476 (from pestis provenance in Tianjun county), pestis phage 087 (from pestis provenance in Tianjun county), pestis phage 072204 (from pestis provenance in same de county); plague phages (from the laboratory of plague bacteria).
3. Experimental methods
(1) Purification method
1) Preparing a bacterial liquid: vaccine strain EV of plague76100 mul is inoculated in 5ml LB liquid culture medium, and is placed in an air bath shaking incubator at 28 ℃ for 18-20h to logarithmic phase, and the strain is taken as a host bacterium.
2) Culturing wild plague phage: respectively taking 1000 μ l of 3 wild type plague phages, respectively adding 5ml LB liquid culture medium, and adding the plague vaccine strain E in logarithmic growth phase into each tube of culture solutionV76Culturing the bacterial suspension 100 μ l and plague phage 2 parts, respectively culturing at 28 deg.C and 37 deg.C for 18-20h, observing optical density of the culture solution the next day, filtering with 0.22 μm filter, and collecting filtrate.
3) Solid culture of wild type plague phage: adding 900 mul LB liquid culture medium into each 2ml EP tube, 10 tubes in total; diluting 3 strains of plague bacteriophage by 10 times respectively, namely respectively sucking 100 μ l of the prepared wild plague bacteriophage, uniformly mixing in a first tube, adding 100 μ l of the prepared wild plague bacteriophage into a second tube, uniformly mixing, and sequentially diluting by 10 times until reaching a 10 th tube; mixing 100 mul of the diluent in each tube with 100 mul of plague vaccine bacterial suspension in logarithmic phase, standing at room temperature for 5min, adding the mixture into melted LB semisolid culture medium with the temperature of 45 ℃ and 6ml, pouring the mixture into the LB solid culture medium at the bottom layer after slight shaking, placing the mixture in a constant-temperature incubator with the temperature of 28 ℃, and observing plaques after 20-24 h.
4) Single-spot culture: single spots of each phage were picked and stored in 1ml of SM solution at 4 ℃. After the phage suspension is diluted by 10 times in series, the plaque is separated according to the double-layer agar plate method, and the process is repeated for 3 times to obtain the purified plague phage.
4. Results of the experiment
The clear brightness of the lysates of No. 087 and No. 072204 is found to be worse than that of No. 476 in the experiment (figure 1); observing the cracking conditions at 28 ℃ and 37 ℃ by adopting a dropping method, and finding that only No. 476 has spots at 28 ℃, No. 087 and No. 072204 have no spots (shown in figure 2); spots appeared at 37 ℃ in 476 and 087, and no spot appeared in 072204 (FIG. 3). The OmniLog TM system therefore selects an intermediate temperature of 33 ℃ as the experimental temperature.
In the single-spot culture, because the natural environment condition is changed into the laboratory environment condition, plague phages 087 and 072204 only have opaque single spots or no spots in the purification process at 37 ℃, and the culture and proliferation are carried out for multiple times by adopting a liquid culture method. Wild-type plague phage 476 were purified 3 times to form plaques of uniform size (see figure 9 for details).
Example 2
A method for identifying plague phage host spectra based on a micro liquid culture medium comprises the following specific steps:
and 7, sealing the plate openings of the 96-hole micro-culture plates, placing the plates in an OmniLogTM system, operating at 33 ℃ for 48h for reading, and observing a kinetic graph of the phenotypic reaction of each group, thereby quantitatively determining the strength of the phenotypic reaction.
The method for identifying the plague phage host spectrum based on the micro liquid culture medium uses the culture medium with small liquid amount, each hole is only 90ul, and the total amount is 100ul by adding the tested bacteria. High throughput assays in which multiple phage interactions with the bacteria being tested can be monitored simultaneously in 1 96-well plate. Adding tetrazole dyes into the culture solution, and automatically measuring the growth condition of the bacteria by the mixed solution of the phage and the tested bacteria through the design of an OmniLog TM system, namely, by relying on redox chemistry and adopting cell respiration as a universal reporter and through the color change of the tetrazole dyes. If the bacteria grow strongly positive, the cells will respire actively, reducing the tetrazolium-based dye and developing a strong color. When phage infection lyses the bacteria, respiration slows or stops, and color is reduced or no color is formed. In addition, a kinetic graph of the phenotypic reaction is finally obtained through an OmniLog TM system, so that the strength of the phenotypic reaction is quantitatively determined. Usually, the curve chart of the action of the mixed solution of the phage added in the first row and the tested bacteria is a horizontal line, and the peak value does not exceed 100, so that the test phage can be judged to infect the added test strain.
The results show that: the curve in the first column is a transverse line, and host bacteria sequentially present different S-shaped curves in each subsequent hole according to the reduction of plague phage titer; the color of the tetrazolium dye in the experimental hole is gradually deepened along with the reduction of the number of the phage and the increase of the number of the host bacteria, and then the plague phage can infect the test bacteria, namely a positive result. The twelve holes in each row present the same S-shaped curve, and the plague phage cannot infect the test strain, which is a negative result. The above results can be used as a basis for potency monitoring of biological properties. If only the presence of plague phages was detected in the samples, only two wells were made, plus blank and negative controls (see figures 5-8 for details).
In the figure, A, B, C, D, E, F rows represent the plague vaccine strains EV respectively76The bacillus subtilis comprises virulent plague bacterium 614F, yersinia pseudotuberculosis PTB3, yersinia pseudotuberculosis PTB5, escherichia coli V517 and enterocolitis bacteria 52302-2, wherein a plague vaccine strain EV76The attenuated plague bacterium 614F is cracked by 4 strains of plague phage; yersinia pseudotuberculosis PTB3, Yersinia pseudotuberculosis PTB5, Escherichia coli V517, enterocolitis bacteria 52302-2 were not lysed by 4 strains of plague phage. Row G as a blank representing fresh LB broth (with tetrazolium-based dye included); rows H1-4 wells served as negative controls and represent 4 strains of plague phage.
Each 96-well plate represents 1 plague phage to be detected, columns 1, 2, 3, 4 and 5 respectively represent different dilution gradients of the plague phage to be detected, and column 6 represents a strain control group in a corresponding row. Each 96-well microplate in fig. 5, 6, 7, 8 represents wild- type plague phages 476, 087, 072204, laboratory diagnostic plague phages, respectively.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for identifying plague phage host spectra based on a micro liquid culture medium is characterized by comprising the following specific steps:
step 1, purifying the separated wild plague bacteriophage, proliferating the wild plague bacteriophage, and selecting a liquid detection method or a semi-solid culture method to measure the titer according to the growth condition of the wild plague bacteriophage to ensure that the titer reaches 106The above steps are reserved;
step 2, selecting experimental plague/non-plague bacteria to be used as host bacteria, hooking lawn in a fresh LB liquid culture medium, placing in an air bath oscillation incubator at 28 ℃, and carrying out oscillation culture at 120r/min for 18-20h to logarithmic phase for later use; adding tetrazole dyes into the bacterial suspension before adding the bacteria-free 96-hole micro-culture plate to ensure that the final concentration of the tetrazole dyes is 20%;
step 3, respectively adding 100ul of wild type plague phages proliferated in the step 1 into the 1 st row of wells of the sterile 96-well microplate, and freely and accurately adding 90ul of LB liquid culture medium into each of the 2 nd to 5 th rows of wells for dilution according to titer of the wild type plague phages;
step 4, respectively taking 10ul of wild plague bacteriophages from the test hole in the row 1, adding the wild plague bacteriophages into the test hole in the row 2, diluting the wild plague bacteriophages by 10 times by using an LB liquid culture medium as a diluent, sequentially diluting the wild plague bacteriophages to the test hole in the row 5 by the same times, uniformly mixing the liquid in the test hole in the row 5, and then discarding 10ul of the diluent;
step 5, taking 10ul of the bacterial suspension containing the tetrazolium dye in the step 2, and respectively adding the bacterial suspension into the 1 st to 6 th columns of test holes in each row of the sterile 96-hole microplate, wherein 1 strain of experimental plague bacillus or non-plague bacillus is added in each row;
step 6, setting the row 1 as a blank control group, adding 98ul of fresh LB liquid culture medium into each test column hole, and then adding 2ul of tetrazole dyes; additionally setting 4 test wells in row 1 as negative control, sequentially adding 3 wild plague bacteriophages to be detected and 1 plague bacteriophage for laboratory diagnosis, and adding 98ul of plague bacteriophages and 2ul of tetrazolium dye into each test well;
and 7, sealing the plate openings of the 96-hole micro-culture plates, placing the plates in an OmniLogTM system, operating at 33 ℃ for 48h for reading, and observing a kinetic graph of the phenotypic reaction of each group, thereby quantitatively determining the strength of the phenotypic reaction.
2. The method for identifying plague phage host profiles based on micro-liquid culture medium according to claim 1, wherein in step 5, the experimental plague bacteria or non-plague bacteria added in each row are sequentially plague vaccine strains EV76The bacillus subtilis comprises virulent plague bacillus 614F, yersinia pseudotuberculosis PTB3, yersinia pseudotuberculosis PTB5, escherichia coli V517 and enterocolitis bacteria 52302-2.
3. The method for identifying a host spectrum of plague phages based on a micro-broth according to claim 1, characterized in that in step 6, the wild type plague phages to be tested are 476 wild type plague phages and 087 wild type plague phages from plague areas in heaven county; wild type plague phage from 072204 plague provenance of the same country; the plague bacteriophage for laboratory diagnosis is from a professional laboratory of plague bacteria.
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