AU2020100857A4 - Method for in vitro screening in-vivo antibacterial activity phages - Google Patents
Method for in vitro screening in-vivo antibacterial activity phages Download PDFInfo
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Abstract
Austracy
The present invention discloses a method for in vitro screening in-vivo
antibacterial activity phages. The method includes the following steps: SI, selecting
an appropriate culture dish, and sterilizing the culture dish; S2, adding a culture
medium into the culture dish, wherein the culture medium includes 10-15 g of beef
soup, 5-10 g of peptone, 30-50 g of sodium chloride, 10-20 g of glucose and 5-18 g
of blood, and mixing the substances to prepare fluid, semisolid and solid culture
media; S3, obtaining serum from experiment subjects, dividing the serum into four
parts, without performing any treatment on the serum A and B, only inactivating the
serum C, and adding a sterilizing agent into the serum D; S4, collecting secretions of
infected persons discharged in vitro from the body, wherein the secretions
specifically include blood, urine, feces, pus or other secretions; S5, selecting
colonies in the secretions, and selecting host bacteria with phages; and S6,
respectively performing following treatments on the four parts of serum: still not
performing any treatment on the serum A, and adding the host bacteria with the
phages into the serum B, the serum C and the serum D. The present invention is high
in screening accuracy.
1
Description
Austracy
The present invention discloses a method for in vitro screening in-vivo antibacterial activity phages. The method includes the following steps: SI, selecting an appropriate culture dish, and sterilizing the culture dish; S2, adding a culture medium into the culture dish, wherein the culture medium includes 10-15 g of beef soup, 5-10 g of peptone, 30-50 g of sodium chloride, 10-20 g of glucose and 5-18 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding a sterilizing agent into the serum D; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; and S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D. The present invention is high in screening accuracy.
Description
Technical Field The present invention relates to the technical field of in-vitro screening of in-vivo antibacterial activity phages, and particularly relates to a method for in vitro screening in-vivo antibacterial activity phages. Background For two existing classical screening techniques, mainly two classical methods for screening phages expressing specific antibodies from a phage antibody library exist. The methods are as follows: pure antigens are coated on solid-phase media, such as ELISA plates, immuno tubes or affinity columns; then to-be-screened phages are added; non-affinity or low-affinity phages are washed away; high-affinity phages are recovered; the antigens are connected with biotin groups; and the antigens are fixed on coated paramagnetic beads to screen the phages. In the two methods, skimmed milk need to be added, effects of the methods are poor, and sites occupied by the antigens are closed, to avoid nonspecific binding of the phages. For the former method, when the phages in specific binding with the antigens are recovered, the phages may be eluted with alkaline solutions such as triethylanmine or acid solutions such as glycine monohydrochloric acid, or eluted with soluble antigens or haptens; while for the latter method, the phages are eluted with dithiothreitol by breaking disulfide bonds between the antigens and biotins. The recovered phages infect host bacteria, and are subjected to next round of screening after proliferation. Generally, by virtue of such a round of screening, cloning expressing high-affinity target antibodies may be obtained. Preconditions of the classical screening techniques are as follows: properties of antigens specified to the target antibodies are clear, and pure antigens can be obtained. With respect to the condition that the antigens cannot be purified or the properties of the antigens are not determined, such as cancer cell surface receptors,
Description
or antigen inactivation in the classical screening process, e.g., intrinsic proteins of certain membranes, the classical screening techniques are not applicable any more. A reference document with an application publication number of CN201310498522.4 discloses a method for in vitro screening in-vivo antibacterial activity phages. According to the method, all types of bacteria and phages related to the bacteria are separated from clinical microbiological test specimens (such as lavage fluid, wounds, feces, urine and the like) beyond sputum specimens of infected persons, but multiple other kinds of bacteria exist in the specimens collected from the infected persons in vitro, and adverse effects are easily achieved to experimental results, thereby further causing phage depletion when the screened phages are affected by other bacteria or cells, and decreasing screening accuracy.
Summary A technical problem to be solved in the present invention is to provide a method for in vitro screening in-vivo antibacterial activity phages with high screening accuracy. To solve the above technical problem, technical solutions of the present invention are provided as follows: the method for in vitro screening in-vivo antibacterial activity phages includes the following steps: S, selecting an appropriate culture dish, and sterilizing the culture dish; S2, adding a culture medium into the culture dish, wherein the culture medium includes 10-15 g of beef soup, 5-10 g of peptone, 30-50 g of sodium chloride, 10-20 g of glucose and 5-18 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding a sterilizing agent into the serum D;
Description
S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum, wherein if growth rates of the host bacteria in the serum B, C and D are consistent, and the growth rates are lower than growth rates of the host bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated. Preferably, in the S1, the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization. Preferably, in the S2, the culture medium includes the following components: g of beef soup, 8 g of peptone, 50 g of sodium chloride, 12 g of glucose and 6 g of blood. Preferably, in the S2, the culture medium includes the following components: g of beef soup, 7 g of peptone, 30 g of sodium chloride, 20 g of glucose and 18 g of blood. Preferably, in the S3, the sterilizing agent is a photocatalyst, and the photocatalyst is used for preparing bismuthyl iodide (BiOI) by a hydrothermal method and preparing titanium dioxide (TiO 2) by a sol-gel method. By adding the photocatalyst, phages resistant to the photocatalyst are screened, thereby eliminating interference of other bacteria and increasing the screening accuracy.
Description
Preferably, in the S3, the sterilizing agent is an inorganic antibacterial agent. By adding the inorganic antibacterial agent, phages resistant to the inorganic antibacterial agent are screened so as to adaptively select the needed phages in experiments, thereby eliminating other interference factors and increasing the screening accuracy. Preferably, in the S3, the sterilizing agent is an organic antibacterial agent. By adding the organic antibacterial agent, phages resistant to the organic antibacterial agent are screened so as to adaptively select the needed phages in experiments, thereby eliminating the other interference factors and increasing the screening accuracy. Preferably, in the S3, the serum C is subjected to inactivation treatment at -50°C for 30 min. Preferably, in the S8, the host bacteria with the phages are counted by using a bacteria counter. Compared with the prior art, the present invention has beneficial effects as follows: By adding the corresponding sterilizing agents, the phages resistant to the sterilizing agents are screened so as to adaptively select the needed phages in the experiments, thereby eliminating the other interference factors and increasing the screening accuracy.
Detailed Description Terms "first" and "second" are used for the purposes of illustration only, but cannot be understood to indicate or imply relative importance or implicitly indicate the quantity of indicated technical feature. Thus, features defined with "first" and "second" may include one or more features clearly or implicitly. In illustration of the present application, a term "multiple" refers to two or more than two, unless otherwise specified. Embodiment 1
Description
The present embodiment discloses a method for in vitro screening in-vivo antibacterial activity phages, including the following steps: S, selecting an appropriate culture dish, and sterilizing the culture dish, wherein the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization; S2, adding a culture medium into the culture dish, wherein the culture medium specifically includes 10 g of beef soup, 8 g of peptone, 50 g of sodium chloride, 12 g of glucose and 6 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding a photocatalyst into the serum D, wherein the photocatalyst is used for preparing bismuthyl iodide (BiOI) by a hydrothermal method and preparing titanium dioxide (TiO 2) by a sol-gel method; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum by using a bacteria counter, wherein if growth rates of the host bacteria in the serum B, C and D are consistent, and the growth rates are lower than growth rates of the host bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated.
Description
By adding the photocatalyst, phages resistant to the photocatalyst are screened, thereby eliminating interference of other bacteria and increasing the screening accuracy. Embodiment 2 S, selecting an appropriate culture dish, and sterilizing the culture dish, wherein the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization; S2, adding a culture medium into the culture dish, wherein the culture medium specifically includes 15 g of beef soup, 7 g of peptone, 30 g of sodium chloride, 20 g of glucose and 18 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding a photocatalyst into the serum D, wherein the photocatalyst is used for preparing bismuthyl iodide (BiOI) by a hydrothermal method and preparing titanium dioxide (TiO 2) by a sol-gel method; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum by using a bacteria counter, wherein if growth rates of the host bacteria in the serum B, C and D are consistent, and the growth rates are lower than growth rates of the host
Description
bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated. The composition proportion of the culture media is changed to observe whether the number of phage extraction is changed, so as to further increase the experiment accuracy. Embodiment 3 S, selecting an appropriate culture dish, and sterilizing the culture dish, wherein the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization; S2, adding a culture medium into the culture dish, wherein the culture medium specifically includes 10 g of beef soup, 8 g of peptone, 50 g of sodium chloride, 12 g of glucose and 6 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding an inorganic antibacterial agent into the serum D; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum by using a bacteria counter, wherein if growth rates of the host bacteria in the serum B, C and D are consistent, and the growth rates are lower than growth rates of the host
'7
Description
bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated. By adding the inorganic antibacterial agent, phages resistant to the inorganic antibacterial agent are screened so as to adaptively select the needed phages in experiments, thereby eliminating other interference factors and increasing the screening accuracy. Embodiment 4 S, selecting an appropriate culture dish, and sterilizing the culture dish, wherein the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization; S2, adding a culture medium into the culture dish, wherein the culture medium specifically includes 10 g of beef soup, 8 g of peptone, 50 g of sodium chloride, 12 g of glucose and 6 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding an organic antibacterial agent into the serum D; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically include blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum by using a bacteria counter, wherein if growth rates of the host bacteria in the serum B,
Description
C and D are consistent, and the growth rates are lower than growth rates of the host bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated. By adding the organic antibacterial agent, phages resistant to the organic antibacterial agent are screened so as to adaptively select the needed phages in experiments, thereby eliminating other interference factors and increasing the screening accuracy. For those skilled in the art, apparently, the present invention is not limited to details of the above exemplary embodiment, and may be realized in other specific forms without deviating from the spirit or essential features of the present invention. Therefore, for every point, the embodiments shall be regarded to be exemplary and non-restrictive. The scope of the present invention is limited by appended claims, rather than the above description. Therefore, all modifications in the meaning and scope of equivalent elements of the claims are included in the present invention. The above embodiments only represent the implementation modes of the present invention. The protection scope of the present invention is not limited to the above embodiments. Several variations and improvements may be made by those skilled in the art without deviating from the concept of the present invention. These variations and improvements belong to the protection scope of the present invention.
Claims (4)
1. A method for in vitro screening in-vivo antibacterial activity phages, comprising the following steps: S, selecting an appropriate culture dish, and sterilizing the culture dish; S2, adding a culture medium into the culture dish, wherein the culture medium comprises 10-15 g of beef soup, 5-10 g of peptone, 30-50 g of sodium chloride, 10-20 g of glucose and 5-18 g of blood, and mixing the substances to prepare fluid, semisolid and solid culture media; S3, obtaining serum from experiment subjects, dividing the serum into four parts, without performing any treatment on the serum A and B, only inactivating the serum C, and adding a sterilizing agent into the serum D, wherein the sterilizing agent is one of a photocatalyst, an organic antibacterial agent and inorganic antibacterial agent, and the photocatalyst is used for preparing bismuthyl iodide (BiOI) by a hydrothermal method and preparing titanium dioxide (TiO2) by a sol-gel method; S4, collecting secretions of infected persons discharged in vitro from the body, wherein the secretions specifically comprise blood, urine, feces, pus or other secretions; S5, selecting colonies in the secretions, and selecting host bacteria with phages; S6, respectively performing following treatments on the four parts of serum: still not performing any treatment on the serum A, and adding the host bacteria with the phages into the serum B, the serum C and the serum D; S7, placing the serum treated in the S5 under light, and regulating an ambient temperature to 37°C; and S8, measuring viable count of the host bacteria in the four parts of serum, wherein if growth rates of the host bacteria in the serum B, C and D are consistent, and the growth rates are lower than growth rates of the host bacteria in the serum A, the to-be-screened phages may serve as candidate phages of animal experiments, otherwise the to-be-screened phages are eliminated.
Claims
2. The method for in vitro screening in-vivo antibacterial activity phages according to claim 1, wherein in the S1, the sterilization measure refers to one of alcohol washing, ultraviolet sterilization or high temperature sterilization.
3. The method for in vitro screening in-vivo antibacterial activity phages according to claim 1, wherein in the S3, the serum C is subjected to inactivation treatment at 45-50°C for 30 min.
4. The method for in vitro screening in-vivo antibacterial activity phages according to claim 1, wherein in the S8, the host bacteria with the phages are counted by using a bacteria counter.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010001658.X | 2020-01-02 | ||
| CN202010001658.XA CN110982793A (en) | 2020-01-02 | 2020-01-02 | Method for in vitro screening of in vivo antibacterial activity phage |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114397443A (en) * | 2021-12-09 | 2022-04-26 | 华南理工大学 | Immunomagnetic bead preservation solution and immunomagnetic bead reagent |
| CN114958607A (en) * | 2022-06-22 | 2022-08-30 | 肖尊平 | Compound microbial agent with high preparation efficiency and preparation method thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112725289A (en) * | 2021-01-25 | 2021-04-30 | 南京悦联生物科技有限公司 | Method for rapidly screening bacteriophage for treatment |
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2020
- 2020-01-02 CN CN202010001658.XA patent/CN110982793A/en not_active Withdrawn
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114397443A (en) * | 2021-12-09 | 2022-04-26 | 华南理工大学 | Immunomagnetic bead preservation solution and immunomagnetic bead reagent |
| CN114958607A (en) * | 2022-06-22 | 2022-08-30 | 肖尊平 | Compound microbial agent with high preparation efficiency and preparation method thereof |
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