CN117305155A - Method for inhibiting salmonella virulence factors - Google Patents
Method for inhibiting salmonella virulence factors Download PDFInfo
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- CN117305155A CN117305155A CN202311090942.9A CN202311090942A CN117305155A CN 117305155 A CN117305155 A CN 117305155A CN 202311090942 A CN202311090942 A CN 202311090942A CN 117305155 A CN117305155 A CN 117305155A
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- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 21
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- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical compound [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a method for inhibiting salmonella virulence factors, which comprises the following steps: inoculating a target salmonella bacteria solution into an improved Martin culture medium for culture to obtain a first bacteria solution; inoculating the first bacterial liquid into a new and improved Martin culture medium for culture to obtain a second bacterial liquid; centrifuging, washing and diluting the second bacterial liquid by adopting an improved Martin solution to obtain bacterial suspension; culturing the bacterial suspension and acetic acid under a certain condition to obtain a third bacterial liquid; and detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid. The detection shows that the expression of salmonella virulence factors can be obviously reduced by acetic acid with different concentrations. The method for inhibiting the salmonella virulence factors can weaken salmonella infection toxicity, so that the infection and incidence probability of salmonella of poultry are reduced, and infection symptoms are relieved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for inhibiting salmonella virulence factors.
Background
Salmonella (Salmonella enterica serovar Enteritidis, SE) is a group of facultative intracellular gram-negative bacteria that are zoonotic, one of the world's important food-borne pathogens, the largest storage host of which is avian. The salmonella enteritidis infection of poultry mainly causes gastroenteritis, and the chicks are infected by means of a faecal route, so that acute infection is caused, higher morbidity and mortality are caused, and huge economic loss is caused for poultry farming; in addition, contaminated poultry and poultry products are a major source of salmonella enteritidis infection in humans, and can transmit pathogens to humans through the food chain, severely threatening human food safety. Therefore, controlling salmonella infection rate is of great importance to both poultry and human health.
Virulence factors refer to specific characteristics or mechanisms possessed by bacteria, viruses, or other pathogens that cause disease in a host. These factors include adhesins, secretory systems, toxins, invasion and biofilm, etc., which increase the ability of pathogens to attack the host, making it easier for pathogens to invade the host, propagate within the host, and cause damage to host tissues. The effects of virulence factors on animals after infection are diverse, for example virulence factors can affect aspects of the host immune system, cellular structure and function, and thus cause serious diseases.
Virulence factors affect the progression of infection and disease by affecting the rate of reproduction and transfer of salmonella, and thus can alleviate symptoms of infection in animals by inhibiting the expression of salmonella virulence factors. Accordingly, it would be desirable to provide a method for inhibiting salmonella virulence factors to reduce salmonella infection toxicity, reduce the infection and incidence of salmonella in birds, and reduce the symptoms of infection, to those skilled in the art.
Disclosure of Invention
The invention provides a method for inhibiting salmonella virulence factors, by which salmonella infection toxicity can be reduced, salmonella infection and incidence probability of poultry can be reduced, and infection symptoms can be relieved.
The technical scheme provided by the invention is as follows:
a method of inhibiting a salmonella virulence factor comprising the steps of:
inoculating a target salmonella bacterial liquid into a first culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain a first bacterial liquid, wherein the first culture medium is an improved Martin culture medium;
inoculating the first bacterial liquid into a second culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain a second bacterial liquid, wherein the second culture medium is an improved Martin culture medium;
collecting the second bacterial liquid, performing a first centrifugation treatment, removing supernatant to obtain a first precipitate, washing the first precipitate with sterile PBS, performing a second centrifugation treatment, collecting a second precipitate, and diluting the second precipitate with an improved Martin solution to obtain bacterial suspension with a certain concentration;
culturing a certain amount of bacterial suspension and acetic acid under a certain condition to obtain a third bacterial liquid;
and detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid.
Further, the method further comprises:
preparing an improved Martin culture medium, and dividing the prepared Martin culture medium into two parts, namely a first culture medium and a second culture medium.
Further, the preparation of the improved martin culture medium comprises the following steps:
28.5g of modified Martin powder was weighed, dissolved in 1000mL of pure water with stirring and heated, and autoclaved at 121℃for 15min to obtain a modified Martin medium.
Further, the inoculation ratio of the target salmonella bacteria liquid to the first culture medium is 1:8-12;
the culture conditions for obtaining the first bacterial liquid are as follows: incubate in a constant temperature shaker at 37℃for 16h at 160 rpm.
Further, the inoculation ratio of the first bacterial liquid to the second culture medium is 1:20-30;
the culture conditions for obtaining the second bacterial liquid are as follows: incubate for 3h at 160rpm in a thermostatted shaker at 37 ℃.
Further, the conditions for performing the first centrifugation and the second centrifugation are as follows: centrifuging at 8000rpm for 5min;
the number of target salmonella in the bacterial suspension is 1×10 7 CFU/mL。
Further, said culturing said bacterial suspension in an amount and under conditions wherein said bacterial suspension is incubated with acetic acid comprises:
taking a certain amount of bacterial suspension, respectively adding the bacterial suspension into a plurality of test tubes, adding a certain amount of acetic acid with different concentrations into all the test tubes, and culturing for a period of time under a certain condition.
Further, a certain amount of the bacterial suspension is taken and respectively added into a plurality of test tubes, then a certain amount of acetic acid with different concentrations is added into all the test tubes, and the bacterial suspension is cultured for a period of time under certain conditions, specifically:
and 5mL of the bacterial suspension is respectively added into 12 test tubes, 5-10mL of acetic acid with different concentrations is added into each test tube, the final concentration of the acetic acid is respectively 0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mmol/L, and the bacterial suspension is cultured for 4 hours at 37 ℃ to obtain third bacterial solutions with different concentrations.
Further, the detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid includes:
180-200L of different third bacterial liquids are respectively added into an ELISA plate, and an ELISA instrument is used for reading a light absorption value at an OD 600 position to detect the growth condition of target salmonella in the different third bacterial liquids;
designing a primer for the target salmonella according to the whole genome sequence of salmonella CMCC50041 and the virulence factor gene sequence in the VFDB database;
and taking different third bacterial liquids, and respectively and sequentially carrying out RNA extraction and RNA reverse transcription, thereby realizing detection of the expression result of the virulence factor of the target salmonella.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for inhibiting salmonella virulence factors, which comprises the steps of inoculating salmonella into an improved Martin culture medium for cultivation twice according to different inoculum sizes, centrifuging to obtain bacterial precipitate, preparing bacterial suspension, and finally culturing the bacterial suspension and acetic acid with different concentrations in vitro, wherein the detection shows that the expression of the salmonella virulence factors can be obviously reduced by the acetic acid with different concentrations. The method can reduce salmonella infection toxicity, thereby reducing infection and incidence probability of salmonella and relieving infection symptoms of poultry.
Drawings
FIG. 1 is a schematic flow chart of a method for inhibiting Salmonella virulence factors according to an embodiment of the invention;
FIG. 2 is a bar graph showing the pH effect of acetic acid at various concentrations on salmonella media cultured in vitro in an example of the present invention;
FIG. 3 is a bar graph showing the effect of acetic acid at various concentrations on salmonella growth in vitro in an example of the present invention;
FIG. 4 is a bar graph showing the effect of different concentrations of acetic acid on the relative expression levels of luxS mRNA in the examples of the present invention;
FIG. 5 is a bar graph showing the effect of acetic acid at various concentrations on the relative expression levels of the sopB mRNA in the examples of the present invention;
FIG. 6 is a bar graph showing the effect of acetic acid at various concentrations on the relative expression levels of csgD mRNA in examples of the present invention;
FIG. 7 is a bar graph showing the effect of acetic acid at various concentrations on the relative expression levels of sipB mRNA in examples of the present invention;
FIG. 8 is a bar graph showing the effect of different concentrations of acetic acid on the relative expression levels of invA mRNA in examples of the present invention;
FIG. 9 is a bar graph showing the effect of acetic acid at various concentrations on the relative expression levels of hilA mRNA in examples of the present invention;
FIG. 10 is a graph showing the comparison of the antibacterial rates of viscera in SE group and acetic acid group in the embodiment of the invention, wherein A is a graph showing the comparison of the antibacterial rate of liver, and B is a graph showing the comparison of the antibacterial rate of spleen;
FIG. 11 is a graph showing the relative expression levels of spleen inflammatory factors TNF-. Alpha.mRNA and IL-1β mRNA in SE and acetate groups according to the example of the present invention;
FIG. 12 is a graph showing the relative expression levels of IL-6mRNA and IL-8mRNA of spleen inflammatory factors in SE group and acetate group in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It will be apparent that the embodiments described below are some, but not all, of the embodiments of the present application. The components of the embodiments of the present application, which are generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations.
Accordingly, the following detailed description of the embodiments of the present application, taken in conjunction with the accompanying drawings, is intended to represent only selected embodiments of the present application, and not to limit the scope of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
Referring to fig. 1, the present invention provides a method for inhibiting salmonella virulence factors, comprising the steps of:
step 1, inoculating a target salmonella bacteria solution into a first culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain the first bacteria solution, wherein the first culture medium is an improved Martin culture medium.
Step 2, inoculating the first bacterial liquid into a second culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain a second bacterial liquid, wherein the second culture medium is an improved Martin culture medium.
Step 3, collecting the second bacterial liquid, performing a first centrifugation treatment, removing supernatant to obtain a first precipitate, washing the first precipitate with sterile PBS, performing a second centrifugation treatment, collecting a second precipitate, and diluting the second precipitate with an improved Martin solution to obtain bacterial suspension with a certain concentration; the preparation method of the improved Martin solution adopted in the step comprises the following steps: 5.0g of peptone, 1.0g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 2.0g of yeast extract powder and 20.0g of glucose, adjusting the pH value to 6.4 and fixing the volume to 1L.
And step 4, taking a certain amount of bacterial suspension and acetic acid to culture under a certain condition, so as to obtain a third bacterial liquid.
And 5, detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid.
Optionally, the method further comprises:
preparing an improved Martin culture medium, and dividing the prepared Martin culture medium into two parts, namely a first culture medium and a second culture medium.
Optionally, the preparation of the improved martin culture medium specifically comprises the following steps:
42.5g of modified Martin powder was weighed, dissolved in 1000mL of pure water with stirring and heated, and autoclaved at 121℃for 15min to obtain a modified Martin medium.
Optionally, the inoculation ratio of the target salmonella bacteria liquid to the first culture medium is 1:8-12;
the culture conditions for obtaining the first bacterial liquid are as follows: incubate in a constant temperature shaker at 37℃for 16h at 160 rpm.
Optionally, the inoculation ratio of the first bacterial liquid to the second culture medium is 1:20-30;
the culture conditions for obtaining the second bacterial liquid are as follows: incubate for 3h at 160rpm in a thermostatted shaker at 37 ℃.
Optionally, the conditions for performing the first centrifugation and the second centrifugation are as follows: centrifuging at 8000rpm for 5min;
the number of target salmonella in the bacterial suspension is 1×10 7 CFU/mL。
Optionally, said culturing said bacterial suspension with acetic acid in an amount and under conditions comprising:
taking a certain amount of bacterial suspension, respectively adding the bacterial suspension into a plurality of test tubes, adding a certain amount of acetic acid with different concentrations into all the test tubes, and culturing for a period of time under a certain condition.
Optionally, a certain amount of the bacterial suspension is taken and added into a plurality of test tubes respectively, then a certain amount of acetic acid with different concentrations is added into all the test tubes, and the bacterial suspension is cultured for a period of time under a certain condition, specifically:
and 5mL of the bacterial suspension is respectively added into 12 test tubes, 5-10mL of acetic acid with different concentrations is added into each test tube, the final concentration of the acetic acid is respectively 0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mmol/L, and the bacterial suspension is cultured for 4 hours at 37 ℃ to obtain third bacterial solutions with different concentrations.
Optionally, the detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid includes:
180-200L of different third bacterial liquids are respectively added into an ELISA plate, and an ELISA instrument is used for reading a light absorption value at an OD 600 position to detect the growth condition of target salmonella in the different third bacterial liquids;
designing a primer for the target salmonella according to the whole genome sequence of salmonella CMCC50041 and the virulence factor gene sequence in the VFDB database;
and taking different third bacterial liquids, and respectively and sequentially carrying out RNA extraction and RNA reverse transcription, thereby realizing detection of the expression result of the virulence factor of the target salmonella.
Example 1
The embodiment provides a method for inhibiting salmonella virulence factors, salmonella is selected from a salmonella standard strain (CMCC 50041), comprising the following steps:
(1) Preparing improved Martin culture medium
42.5g of modified Martin powder is weighed, heated, stirred and dissolved in 1000mL of pure water, and sterilized at 121 ℃ under high pressure for 15min to obtain modified Martin culture medium for later use.
(2) Co-culture of acetic acid and Salmonella
1) Salmonella was removed from the-80 ℃ refrigerator and inoculated into modified martin medium (bacterial liquid: modified Martin medium=1:10, and incubated in a constant temperature shaker at 37℃for 16h at 160rpm, giving a first bacterial solution.
2) The next day, the first bacterial liquid was taken out and inoculated into another modified Martin medium (bacterial liquid: modified martin medium=1:25), and incubated at 160rpm in a constant temperature shaker at 37℃for 3h to give a second bacterial solution.
3) The second bacterial solution was collected, centrifuged at 8000rpm for 5min, the supernatant was discarded, the pellet was washed once with sterile PBS, centrifuged at 8000rpm for 5min, and the bacterial pellet was collected. Diluting the bacterial pellet with modified Martin until Salmonella number is 1×10 7 CFU/mL to obtain bacterial suspension of salmonella, and storing in a refrigerator at 4 ℃ for later use.
4) Taking 12 test tubes, respectively adding 5mL of bacterial suspension, adding 5-10mL of acetic acid with different concentrations into each test tube, so that the final concentration of the acetic acid is 0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mmol/L respectively, and culturing for 4 hours at 37 ℃ to obtain a final culture solution.
And then, respectively carrying out PH detection, salmonella growth condition detection and salmonella virulence factor expression detection on the final culture solution.
Example two
1. Effect of acetic acid on the pH of the in vitro culture Salmonella Medium
1) The pH meter was calibrated with buffer, and the electrodes were immersed in standard buffer mixed with phosphate standard buffer (6.86) and stirred well. When the value is stable and S appears, the STANDARDIZE key is pressed, and the automatic calibration of the instrument is waited. After calibration, "6.86" and electrode slope are displayed. The electrode is taken out from the buffer solution, and after the electrode is cleaned, the electrode is immersed into the potassium hydrogen phthalate standard buffer solution (4.01) and stirred uniformly. When the value reaches to be stable and 'S' appears, the 'STANDARDIZE' key is pressed, and the automatic calibration of the instrument is waited. After calibration, the values are stored as second calibration points, showing (4.01, 6.86) and electrode slope. The third point (boric acid solution, 9.18) correction was completed in the same procedure. After the correction, the electrodes were immersed in a 4M KCl solution and stored.
2) And cleaning the electrodes, immersing the electrodes into the final culture solution respectively, and reading after the values are stable. As shown in FIG. 2, it was found that acetic acid at a concentration of 1.56mmol/L or less had no effect on the pH of the medium.
2. Effect of acetic acid on growth of Salmonella in vitro culture
And respectively sucking 180-200L of bacterial liquid from 12 final culture liquids, adding the bacterial liquid into an ELISA plate, reading a light absorption value at an OD 600 by using an ELISA instrument, and detecting bacterial growth. As shown in FIG. 3, it was found that the concentration of acetic acid at 1.56mmol/L or less had no effect on the growth of bacteria.
3. Effect of acetic acid on Salmonella virulence factor expression
(1) Primer design
The design synthesis was performed based on the whole genome sequence of salmonella CMCC50041 and the virulence factor gene sequence in the VFDB database (as shown in table 1).
TABLE 1 Salmonella virulence factor primers
(2) Extraction of RNA
Collecting the final culture solution, centrifuging at 12000rpm for 5min, discarding supernatant, collecting bacterial precipitate, re-suspending bacterial precipitate with 1×PBS, centrifuging again, discarding supernatant; bacterial pellet was resuspended in 2mL PBS, 500. Mu.l RNA-easy was added and the pellet was repeatedly pipetted until well lysed; 200 μl RNase-free dd H was added to the lysates 2 O, mixing the materials upside down, and standing at room temperature for 5min; centrifuging at 12000rpm at room temperature for 15min, taking out the centrifuge tube, and carefully sucking the upper water phase into a new centrifuge tube; adding isopropyl alcohol with equal volume, mixing upside down, standing at room temperature for 10min; centrifuging at 12000rpm at room temperature for 10min, precipitating white at the bottom of the centrifuge tube, and discarding supernatant; adding 500 mu L of 75% ethanol, suspending and precipitating, and reversing for several times; centrifuging at 8000rpm at room temperature for 3min, and discarding supernatant; repeating the step of washing and precipitating with 75% ethanol; air-dried at room temperature, and 50. Mu.l of RNase-free ddH was added 2 O was dissolved and precipitated, and the RNA was sufficiently dissolved by repeated pipetting with a pipette.
(3) RNA reverse transcription
1) Genomic DNA removal
Preparing mixed solution
2) Preparation of reverse transcription reaction System
Directly adding 5X HiScript III qRT SuperMix into a reaction tube
3) Reverse transcription reaction
(4) mRNA expression level detection
mRNA level detection was performed on the cDNA obtained in 3), and the reaction system was as follows:
1) Preparing mixed solution
2) qPCR reaction
qPCR results show that acetic acid at different concentrations can significantly reduce the expression of salmonella virulence factors (as shown in FIGS. 4-9).
Example III
96 SPF chickens with the weight close to 1 day old are selected and randomly divided into 2 treatment groups (SE group and acetic acid group), 24 chickens in each treatment group are fed into 2 SPF sterile cabins, basic daily ration is fed, and 1% acetic acid is added into drinking water in the acetic acid group. Salmonella subcutaneously injected 10 in all chickens at 7 days of age 8 CFU/animal, 8 day old slaughter, and collect samples. The results of visceral bacterial load showed that the liver and spleen bacterial load of the acetic acid group were significantly lower than that of the SE group (shown in FIG. 10), and that the spleen inflammatory responses (TNF-alpha, IL-1β, IL-6 and IL-8) caused by SE were also relieved, as shown in FIGS. 11 and 12.
In summary, the invention provides a method for inhibiting salmonella virulence factors, which can effectively inhibit the expression of salmonella virulence factors by co-culturing salmonella, and weaken salmonella infection toxicity by the method, thereby reducing the infection and incidence probability of salmonella and alleviating infection symptoms of poultry.
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (9)
1. A method of inhibiting a salmonella virulence factor comprising the steps of:
inoculating a target salmonella bacterial liquid into a first culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain a first bacterial liquid, wherein the first culture medium is an improved Martin culture medium;
inoculating the first bacterial liquid into a second culture medium according to a certain proportion, and culturing for a period of time under a certain condition to obtain a second bacterial liquid, wherein the second culture medium is an improved Martin culture medium;
collecting the second bacterial liquid, performing a first centrifugation treatment, removing supernatant to obtain a first precipitate, washing the first precipitate with sterile PBS, performing a second centrifugation treatment, collecting a second precipitate, and diluting the second precipitate with an improved Martin solution to obtain bacterial suspension with a certain concentration;
culturing a certain amount of bacterial suspension and acetic acid under a certain condition to obtain a third bacterial liquid;
and detecting the expression of the virulence factor of the target salmonella in the third bacterial liquid.
2. The method of inhibiting a salmonella virulence factor of claim 1, further comprising:
preparing an improved Martin culture medium, and dividing the prepared Martin culture medium into two parts, namely a first culture medium and a second culture medium.
3. The method for inhibiting salmonella virulence factors according to claim 2, wherein the preparation of the modified martin medium comprises:
28.5g of modified Martin powder was weighed, dissolved in 1000mL of pure water with stirring and heated, and autoclaved at 121℃for 15min to obtain a modified Martin medium.
4. The method of inhibiting a salmonella virulence factor of claim 1, wherein:
the inoculation ratio of the target salmonella bacteria liquid to the first culture medium is 1:8-12;
the culture conditions for obtaining the first bacterial liquid are as follows: incubate in a constant temperature shaker at 37℃for 16h at 160 rpm.
5. The method of inhibiting a salmonella virulence factor of claim 1, wherein:
the inoculation ratio of the first bacterial liquid to the second culture medium is 1:20-30;
the culture conditions for obtaining the second bacterial liquid are as follows: incubate for 3h at 160rpm in a thermostatted shaker at 37 ℃.
6. The method of inhibiting a salmonella virulence factor of claim 1, wherein:
the conditions for carrying out the first centrifugal treatment and the second centrifugal treatment are as follows: centrifuging at 8000rpm for 5min;
the number of target salmonella in the bacterial suspension is 1×10 7 CFU/mL。
7. The method of inhibiting a virulence factor of salmonella of any one of claims 1-6, wherein the culturing of the bacterial suspension with acetic acid in an amount is performed under conditions comprising:
taking a certain amount of bacterial suspension, respectively adding the bacterial suspension into a plurality of test tubes, adding a certain amount of acetic acid with different concentrations into all the test tubes, and culturing for a period of time under a certain condition.
8. The method of claim 7, wherein the bacterial suspension is added to a plurality of test tubes, and acetic acid with different concentrations is added to all test tubes, and the bacterial suspension is cultured for a period of time under certain conditions, specifically:
and 5mL of the bacterial suspension is respectively added into 12 test tubes, 5-10mL of acetic acid with different concentrations is added into each test tube, the final concentration of the acetic acid is respectively 0, 0.1, 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100mmol/L, and the bacterial suspension is cultured for 4 hours at 37 ℃ to obtain third bacterial solutions with different concentrations.
9. The method of claim 8, wherein detecting the expression of the virulence factor of the target salmonella in the third bacterial fluid comprises:
180-200L of different third bacterial liquids are respectively added into an ELISA plate, and an ELISA instrument is used for reading a light absorption value at an OD 600 position to detect the growth condition of target salmonella in the different third bacterial liquids;
designing a primer for the target salmonella according to the whole genome sequence of salmonella CMCC50041 and the virulence factor gene sequence in the VFDB database;
and taking different third bacterial liquids, and respectively and sequentially carrying out RNA extraction and RNA reverse transcription, thereby realizing detection of the expression result of the virulence factor of the target salmonella.
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Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999013053A1 (en) * | 1997-09-10 | 1999-03-18 | Vion Pharmaceuticals, Inc. | Genetically modified tumor-targeted bacteria with reduced virulence |
| WO2000004919A2 (en) * | 1998-07-24 | 2000-02-03 | Megan Health, Inc. | Live attenuated salmonella vaccines to control avian pathogens |
| WO2000032152A2 (en) * | 1998-12-02 | 2000-06-08 | Princeton University | Compositions and methods for regulating bacterial pathogenesis |
| US20020072052A1 (en) * | 1998-12-02 | 2002-06-13 | Bassler Bonnie L. | Compositions and methods for regulating bacterial pathogenesis |
| US20040033548A1 (en) * | 1998-12-02 | 2004-02-19 | Bassler Bonnie L. | Compositions and methods for regulating bacterial pathogenesis |
| JP2004161620A (en) * | 2002-11-11 | 2004-06-10 | Yoshikazu Iwakiri | Prophylactic/therapeutic agent for salmonella infectious disease of animal |
| WO2005024005A1 (en) * | 2003-09-03 | 2005-03-17 | Intralytix, Inc. | Novel salmonella bacteriophage and uses thereof |
| RU2007139681A (en) * | 2007-10-29 | 2009-05-10 | Общество с ограниченной ответственностью "Амфита" (ООО "Амфита") (RU) | IMMUNOBIOLOGICAL BACTERICIDAL DRUG (OPTIONS) |
| WO2010033798A2 (en) * | 2008-09-18 | 2010-03-25 | Aviex Technologies Llc | Live bacterial vaccines resistant to carbon dioxide (co2), acidic ph and/or osmolarity for viral infection prophylaxis or treatment |
| CN101962625A (en) * | 2010-04-29 | 2011-02-02 | 河南科技大学 | Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof |
| US20120276054A1 (en) * | 2011-04-28 | 2012-11-01 | Williams Henry N | Alternative bacterial treatment |
| CN105248932A (en) * | 2015-08-25 | 2016-01-20 | 四川农业大学 | Trace element premix capable of reducing propagation and invasion of Salmonella Enteritidis phage type 4 in laying hen and preparation method thereof |
| US20210338637A1 (en) * | 2015-07-07 | 2021-11-04 | Life Matters Ltd. | Indole derivatives for biofilm disruption and inhibition |
| CN116286671A (en) * | 2023-01-09 | 2023-06-23 | 青岛诺安百特生物技术有限公司 | Salmonella phage SP8, phage composition and application thereof |
-
2023
- 2023-08-28 CN CN202311090942.9A patent/CN117305155B/en active Active
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999013053A1 (en) * | 1997-09-10 | 1999-03-18 | Vion Pharmaceuticals, Inc. | Genetically modified tumor-targeted bacteria with reduced virulence |
| WO2000004919A2 (en) * | 1998-07-24 | 2000-02-03 | Megan Health, Inc. | Live attenuated salmonella vaccines to control avian pathogens |
| CN1315871A (en) * | 1998-07-24 | 2001-10-03 | 梅根保健股份有限公司 | Live attenuated i(salmonella) vaccines to control avian pathogens |
| WO2000032152A2 (en) * | 1998-12-02 | 2000-06-08 | Princeton University | Compositions and methods for regulating bacterial pathogenesis |
| US20020072052A1 (en) * | 1998-12-02 | 2002-06-13 | Bassler Bonnie L. | Compositions and methods for regulating bacterial pathogenesis |
| US20040033548A1 (en) * | 1998-12-02 | 2004-02-19 | Bassler Bonnie L. | Compositions and methods for regulating bacterial pathogenesis |
| JP2004161620A (en) * | 2002-11-11 | 2004-06-10 | Yoshikazu Iwakiri | Prophylactic/therapeutic agent for salmonella infectious disease of animal |
| WO2005024005A1 (en) * | 2003-09-03 | 2005-03-17 | Intralytix, Inc. | Novel salmonella bacteriophage and uses thereof |
| RU2007139681A (en) * | 2007-10-29 | 2009-05-10 | Общество с ограниченной ответственностью "Амфита" (ООО "Амфита") (RU) | IMMUNOBIOLOGICAL BACTERICIDAL DRUG (OPTIONS) |
| WO2010033798A2 (en) * | 2008-09-18 | 2010-03-25 | Aviex Technologies Llc | Live bacterial vaccines resistant to carbon dioxide (co2), acidic ph and/or osmolarity for viral infection prophylaxis or treatment |
| CN101962625A (en) * | 2010-04-29 | 2011-02-02 | 河南科技大学 | Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof |
| US20120276054A1 (en) * | 2011-04-28 | 2012-11-01 | Williams Henry N | Alternative bacterial treatment |
| US20210338637A1 (en) * | 2015-07-07 | 2021-11-04 | Life Matters Ltd. | Indole derivatives for biofilm disruption and inhibition |
| CN105248932A (en) * | 2015-08-25 | 2016-01-20 | 四川农业大学 | Trace element premix capable of reducing propagation and invasion of Salmonella Enteritidis phage type 4 in laying hen and preparation method thereof |
| CN116286671A (en) * | 2023-01-09 | 2023-06-23 | 青岛诺安百特生物技术有限公司 | Salmonella phage SP8, phage composition and application thereof |
Non-Patent Citations (13)
| Title |
|---|
| AMIN N. OLAIMAT等: "The Use of Malic and Acetic Acids in Washing Solution to Control Salmonella spp. on Chicken Breast", JOURNAL OF FOOD SCIENCE, vol. 83, no. 8, 27 July 2018 (2018-07-27), pages 2197 - 2203 * |
| SARA D. LAWHON等: "Intestinal short-chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA", MOLECULAR MICROBIOLOGY, vol. 46, no. 5, 9 December 2002 (2002-12-09), pages 1451 - 1464 * |
| 余荣;徐小芳;王雯熙;: "丁酸对动物肠道影响的研究进展", 中国畜牧杂志, no. 16, 25 August 2012 (2012-08-25), pages 64 - 68 * |
| 俞滨;: "降低家禽养殖业沙门氏菌感染的研究进展", 中国饲料, no. 10, 20 May 2019 (2019-05-20), pages 12 - 16 * |
| 刘杰;韩薇;崔雪志;吴蕾;常海霞;韩帅琪;张向奎;刘振林;李文锋;李建和;李超;许传田;: "常见消毒剂消毒效果评价", 家禽科学, no. 09, 25 September 2020 (2020-09-25), pages 3 - 7 * |
| 吴光先: "用过氧乙酸消除光鸡体表带染沙门氏菌的试验", 畜牧与兽医, no. 05, 30 September 1980 (1980-09-30), pages 12 - 14 * |
| 张婷婷;陈健;周岩民;: "沙门氏菌在肉鸡生产中的危害及其饲料控制技术", 家畜生态学报, no. 06, 15 November 2010 (2010-11-15), pages 113 - 116 * |
| 徐引弟;郭爱珍;陈焕春;: "减毒猪霍乱沙门氏菌作为疫苗及载体的研究进展", 国外畜牧学(猪与禽), no. 06, 25 November 2007 (2007-11-25), pages 71 - 74 * |
| 梁锦宁;李家富;欧阳轶强;张名媛;孙俊铭;郭晓萍;: "6种常用消毒剂对不同细菌消毒效果的比较", 实验动物科学, no. 01, 28 February 2020 (2020-02-28), pages 38 - 41 * |
| 蒋小强;邱红辉;符乃方;蒋盛军;董志超;何际禅;阚飙;闫梅英;梁未丽;: "红茶菌和中草药红茶菌体外抑制沙门氏菌的比较研究", 安徽农业科学, no. 06, 10 February 2015 (2015-02-10), pages 135 - 137 * |
| 赵景鹏;李培勇;王红玉;宋益贞;孙淑红;林海;: "不同精油与酸化剂组合对肉仔鸡肠炎沙门氏菌感染的控制效果研究", 动物营养学报, no. 07, 13 June 2018 (2018-06-13), pages 2672 - 2682 * |
| 阚刘刚;赵丽杰;李秀业;曾丹;王忠;: "鸡沙门氏菌病的生物预防和控制研究进展", 动物营养学报, no. 09, 31 July 2018 (2018-07-31), pages 3432 - 3443 * |
| 齐立辉;郑新艳;冉婷婷;徐朗莱;王伟武;: "沙门氏菌草酰乙酸脱羧酶在大肠杆菌中的表达、纯化及其生化鉴定", 南京农业大学学报, no. 04, 15 July 2010 (2010-07-15), pages 81 - 86 * |
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