CN104312984A - Method for separating phage in water - Google Patents
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Abstract
The invention discloses a method for separating phage in water. The method comprises the following steps: (1) preparing positive charge silica gel particles; (2) preparing eluent; and (3) separating the phage. According to the method disclosed by the invention, the phage in various water environments such as running water, surface water and sewage can be efficiently separated, the separation efficiency is high, and the separation efficiency on the phage of extremely low concentration (100PFU/100L) in the water is 70 percent. The method has the advantages of simplicity and convenience in operation, rapidness and low price. Therefore, the method has the advantages that the speed of separating novel phages from the water environment is increased, and assistance is provided for application of the phages.
Description
Technical field
The invention belongs to microbe separation technology, particularly relate to a kind of water pnagus medius separation method.
Background technology
Phage is the general name of the bacteriophage of the prokaryotic micro-organisms such as bacterial infection, actinomycetes or mycoplasma, and it is the one of virus.Before the discovery of phage can trace back to more than 100 year, Britain bacteriologist Ernes Hankin reported first in 1896 India's river exists a kind of by porcelain filter, thermolability and there is the material of strong anti-microbial activity, regrettably Hankin could not continue the research of this aspect.Until 1915, Britain microbiologist Frederick Twort has found fenestra first on the substratum scribbling Staphylococcus aureus, and this phenomenon is illustrated as the hypothesis of virus infection further.Canada microbiologist F é lix d ' Herelle is formal in 1917 is phage by this viral nomenclature.Phage structure is simple, and be only made up of the capsid protein of outside and the nucleic acid of inside, its nucleic acid substances can be divided into dsDNA, ssDNA, dsRNA and ssRNA.A salient features of phage is exactly its Host Strains of cracking that can be single-minded, people have found the extensive use of phage with this, mainly comprise the Phage therapy, deactivation food, the biological control of pathogenic bacterium in water and plant and the phage that are used for the treatment of multi-drug resistant bacteria and infect and detect.
Phage is widely distributed at occurring in nature, and almost have the place of bacteria live just to there is corresponding phage, its quantity reaches 10
32, be about 10 times of bacterium.Because phage is to the single-minded cracking of Host Strains, the application of phage is restricted, and people have invented drug cocktail therapy (treatment) for this reason, and this also just needs more how different phages.But up to the present, only have the phage of strain more than 6000 to be found and carried out morphologic description, the phage actual quantity that this and occurring in nature exist also exists huge spread.Along with the widespread use of phage, people need to obtain more phage, but new phage finds that speed but can not meet this active demand far away.
Traditional water pnagus medius separation method for volume of water sample generally only have 1-1000mL, be therefore difficult to be separated to the phage that in water, concentration is lower.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of easy to use, the water pnagus medius separation method of large water sample can be processed.
Technical scheme of the present invention is summarized as follows:
A kind of water pnagus medius separation method, comprises the steps:
(1) preparation of positive charge silica gel particle
1. in proportion, take 6-7g aluminum chloride under room temperature slowly to join in 950mL distilled water and make dissolving; Add 45mL, 2M aqueous sodium carbonate, occur milky Al (OH)
3gel, adjust ph, to 7.0-7.5, adds water and is settled to 1000mL; At room temperature leave standstill 18-24h;
2. liquid step 1. obtained is to abandon supernatant after the centrifugal 10-30min of 1100g; In precipitation, add 0.14M sodium chloride aqueous solution to the resuspended precipitation of 1000mL, leave standstill 18-24h;
3. repeating step 2. 2-4 time, in 121 DEG C of autoclaving 20min, is cooled to normal temperature;
4. getting 1200-1400g particle diameter is that 150-300 μm of silica gel joins in the liquid that 3. step obtain, and limit edged stirs, and makes it abundant mixing, is placed in 50-100 DEG C of baking box and dries, obtain positive charge silica gel particle;
(2) preparation of elutriant
Take 30g Tryptones, 15g beef powder, 15g sodium-chlor, 30-40g glycine and 20-30g sodium hydroxide adding distil water to 1L, heating for dissolving, adjustment pH is 10.2-10.5, autoclaving 20min at 121 DEG C;
(3) separation of phage:
1. in chromatography column, add appropriate sterile purified water, then add positive charge silica gel particle and constantly stir removal bubble, with 300-700mL/min speed, water sample to be measured is flow through chromatography column from the top of chromatography column by peristaltic pump;
2., after water sample to be measured all flows through chromatography column, with being equivalent to positive charge silica gel particle quality 3-4 elution doubly, collect wash-out effluent liquid, adjustment pH is 7.0-7.5; Add PEG6000 make mass concentration be 10%-15% and under agitation make PEG6000 dissolve, leave standstill 20-40min at normal temperatures, the centrifugal 20-40min of 12000-16000g, abandon supernatant liquor, be settled to 10-100mL obtain liquid 1 by the resuspended precipitation of the PBS of 0.1MpH=7.0-7.5, in liquid 1, add 10 × liquid nutrient medium, add that volume is liquid 1 volume 1/10 of described 10 × liquid nutrient medium, obtains liquid 2;
3. separately get 1mL10 × liquid nutrient medium to add sterile saline and obtain 1 × liquid nutrient medium to 10mL, the Host Strains of phage is seeded in 1 × liquid nutrient medium, at 30-37 DEG C, cultivates 18-24h;
4. getting the bacterium liquid that 1mL step (3) 3. obtains is seeded in liquid 2,18-24h is cultivated at 30-37 DEG C, then the centrifugal 10-20min of 3000-4000g, collect supernatant liquor, whether get after bacterium liquid that 1mL supernatant liquor and 100 μ L steps (3) 3. obtain mixes and leave standstill 5min, adopting double-layer agar technique to detect in supernatant liquor has plaque to occur.
The Host Strains of phage is colon bacillus, Salmonellas, colon bacillus O157 ︰ H7, streptococcus aureus, streptococcus faecium, Shigellae, Vibrio parahemolyticus or Yersinia enterocolitica.
Advantage of the present invention:
Method of the present invention can be separated various water surrounding efficiently as the phage in tap water, surface water and sewage, and separation efficiency is high, to the phage (10 of extremely low concentration in water
0pFU/100L) separation efficiency reaches 70%.Present method has the advantages such as easy and simple to handle, quick and cheap, and therefore the method will speed up the speed being separated new phage from water surrounding, for the application of phage is offered help.
Accompanying drawing explanation
Fig. 1 is particulate scan Electronic Speculum figure: a is silica gel particle, b positive charge silica gel particle.
Fig. 2 is silica gel particle and positive charge silica gel particle grain size distribution.
Embodiment
In order to make those skilled in the art better understand technical scheme of the present invention, below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
After testing without colon bacillus phage MS
2tap water (at the present embodiment referred to as tap water) in add colon bacillus phage MS
2separation, comprise the steps:
(1) preparation of positive charge silica gel particle
1. take 6.675g aluminum chloride under room temperature slowly to join in 950mL distilled water and make dissolving; Add 45mL, 2M aqueous sodium carbonate, occur milky Al (OH)
3gel, adjust ph to 7.2, adds water and is settled to 1000mL; At room temperature leave standstill 24h;
2. liquid step 1. obtained is to abandon supernatant after the centrifugal 15min of 1100g; In precipitation, add 0.14M sodium chloride aqueous solution to the resuspended precipitation of 1000mL, leave standstill 24h;
3. repeating step 2. 3 times, in 121 DEG C of autoclaving 20min, is cooled to normal temperature;
4. getting 1375g particle diameter is that 270 μm of silica gel join in the liquid that 3. step obtain, and limit edged stirs, and makes it abundant mixing, is placed in 60 DEG C of baking boxs and dries, obtain positive charge silica gel particle;
(2) preparation of elutriant
Take 30g Tryptones, 15g beef powder, 15g sodium-chlor, 37.5g glycine and 20g sodium hydroxide adding distil water to 1L, heating for dissolving, pH is regulated to be 10.2 with 0.1mol/L hydrochloric acid soln or 0.1mol/L aqueous sodium hydroxide solution, autoclaving 20min at 121 DEG C;
(3) separation of phage:
1. add in 100L tap water Sulfothiorine (adding 0.5mL mass concentration in every premium on currency is 10% sodium thiosulfate solution) with in and chlorine residue in water, then at water sample inoculation 1ml phage MS
2(10
0and fully mix to obtain water sample to be measured PFU/100L);
Internal diameter be 83.5mm, high for the chromatography column of 400mm in add 1.5L sterile purified water, then add 800g positive charge silica gel particle and constantly stir removal bubble, with 500mL/min speed, water sample to be measured is flow through chromatography column by peristaltic pump from the top of chromatography column;
2. after water sample to be measured all flows through chromatography column, with the elution of 2800g, collect wash-out effluent liquid, regulate pH to be 7.2; Add PEG6000 make mass concentration be 13% and under agitation make PEG6000 dissolve, leave standstill 30min at normal temperatures, the centrifugal 30min of 15000g, abandon supernatant liquor, be settled to 50mL obtain liquid 1 by the resuspended precipitation of the PBS of 0.1MpH=7.2, add in liquid 1 5mL10 × LB liquid nutrient medium, obtain liquid 2
3. separately get 1mL10 × LB liquid nutrient medium to add sterile saline and obtain 1 × LB liquid nutrient medium to 10mL, the Host Strains colon bacillus 15597 of phage is seeded in 1 × LB liquid nutrient medium, at 35 DEG C, cultivates 24h;
4. getting the bacterium liquid that 1mL step (3) 3. obtains is seeded in liquid 2,24h is cultivated at 37 DEG C, then the centrifugal 10min of 4000g, collect supernatant liquor, get after bacterium liquid that 1mL supernatant liquor and 100 μ L steps (3) 3. obtain mixes and leave standstill 5min, adopting double-layer agar technique to detect in supernatant liquor has plaque to occur.
Embodiment 2
In river, the separation of colon bacillus 15597 phage, comprises the steps:
(1) preparation of positive charge silica gel particle
1. take 6g aluminum chloride under room temperature slowly to join in 950mL distilled water and make dissolving; Add 45mL, 2M aqueous sodium carbonate, occur milky Al (OH)
3gel, adjust ph to 7.0, adds water and is settled to 1000mL; At room temperature leave standstill 18h;
2. liquid step 1. obtained is to abandon supernatant after the centrifugal 10min of 1100g; In precipitation, add 0.14M sodium chloride aqueous solution to the resuspended precipitation of 1000mL, leave standstill 18h;
3. repeating step 2. 2 times, in 121 DEG C of autoclaving 20min, is cooled to normal temperature;
4. getting 1200g particle diameter is that 150 μm of silica gel join in the liquid that 3. step obtain, and limit edged stirs, and makes it abundant mixing, is placed in 50 DEG C of baking boxs and dries, obtain positive charge silica gel particle;
(2) preparation of elutriant
Take 30g Tryptones, 15g beef powder, 15g sodium-chlor, 30g glycine and 30g sodium hydroxide adding distil water to 1L, heating for dissolving, regulate pH to be 10.5, autoclaving 20min at 121 DEG C;
(3) separation of phage
1. internal diameter be 83.5mm, high for the chromatography column of 400mm in add 1.5L sterile purified water, add 800g positive charge silica gel particle again and constantly stir removal bubble, with 300mL/min speed, water sample river to be measured for 100L is flow through chromatography column from the top of chromatography column by peristaltic pump;
2. after water sample to be measured all flows through chromatography column, with the elution of 2400g, collect wash-out effluent liquid, regulate pH to be 7.0; Adding PEG6000 makes mass concentration be 10%, PEG6000 is under agitation made to dissolve, leave standstill 20min at normal temperatures, the centrifugal 40min of 12000g, abandon supernatant liquor, be settled to 10mL obtain liquid 1 by the resuspended precipitation of the PBS of 0.1MpH=7.0, in liquid 1, add 1mL10 × LB liquid nutrient medium, obtain liquid 2;
3. separately get 1mL10 × LB liquid nutrient medium to add sterile saline and obtain 1 × LB liquid nutrient medium to 10mL, the Host Strains (colon bacillus 15597) of phage is seeded in 1 × LB liquid nutrient medium, at 30 DEG C, cultivates 24h;
4. getting the bacterium liquid that 1mL step (3) 3. obtains is seeded in liquid 2,18h is cultivated at 30 DEG C, then the centrifugal 20min of 3000g, collect supernatant liquor, get after bacterium liquid that 1mL supernatant liquor and 100 μ L steps (3) 3. obtain mixes and leave standstill 5min, adopting double-layer agar technique to detect in supernatant liquor has plaque to occur.
Embodiment 3
In sewage, the separation of colon bacillus 15597 phage, comprises the steps:
(1) preparation of positive charge silica gel particle
1. in proportion, take 7g aluminum chloride under room temperature slowly to join in 950mL distilled water and make dissolving; Add 45mL, 2M aqueous sodium carbonate, occur milky Al (OH) 3 gel, adjust ph to 7.5, adds water and is settled to 1000mL; At room temperature leave standstill 20h;
2. liquid step 1. obtained is to abandon supernatant after the centrifugal 30min of 1100g; In precipitation, add 0.14M sodium chloride aqueous solution to the resuspended precipitation of 1000mL, leave standstill 20h;
3. repeating step 2. 4 times, in 121 DEG C of autoclaving 20min, is cooled to normal temperature;
4. getting 1400g particle diameter is that 300 μm of silica gel join in the liquid that 3. step obtain, and limit edged stirs, and makes it abundant mixing, is placed in 100 DEG C of baking boxs and dries, obtain positive charge silica gel particle;
(2) take 30g Tryptones, 15g beef powder, 15g sodium-chlor, 40g glycine and 20g sodium hydroxide adding distil water to 1L, heating for dissolving, regulate pH to be 10.2, autoclaving 20min at 121 DEG C;
(3) separation of phage:
1. internal diameter be 83.5mm, high for the chromatography column of 400mm in add 1.5L sterile purified water, then add 800g positive charge silica gel particle and constantly stir removal bubble, with 700mL/min speed, 100L sewage is flow through chromatography column by peristaltic pump from the top of chromatography column;
2. after water sample to be measured all flows through chromatography column, use 3200g elution, collect wash-out effluent liquid, regulate pH to be 7.5; Add PEG6000 make mass concentration be 15% and under agitation make PEG6000 dissolve, leave standstill 40min at normal temperatures, the centrifugal 20min of 16000g, abandon supernatant liquor, be settled to 100mL obtain liquid 1 by the resuspended precipitation of the PBS of 0.1MpH=7.5, in liquid 1, add 10mL10 × LB liquid nutrient medium, add that volume is liquid 1 volume 1/10 of described 10 × LB liquid nutrient medium, obtains liquid 2;
3. separately get 1mL10 × LB liquid nutrient medium to add sterile saline and obtain 1 × LB liquid nutrient medium to 10mL, the Host Strains colon bacillus 15597 of phage is seeded in 1 × LB liquid nutrient medium, at 37 DEG C, cultivates 18h;
4. getting the bacterium liquid that 1mL step (3) 3. obtains is seeded in liquid 2,20h is cultivated at 30 DEG C, then the centrifugal 15min of 3000g, collect supernatant liquor, get after bacterium liquid that 1mL supernatant liquor and 100 μ L steps (3) 3. obtain mixes and leave standstill 5min, adopting double-layer agar technique to detect in supernatant liquor has plaque to occur.
Embodiment 4
The separation of Salmonellas 50001 phage in river
By the colon bacillus in Salmonellas 50001 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having Salmonellas 50001 phage occurs.
Embodiment 5
The separation of colon bacillus O157 ︰ H721530 phage in river
By the colon bacillus in colon bacillus O157 ︰ H721530 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having colon bacillus O157 ︰ H721530 phage occurs.
Embodiment 6
The separation of streptococcus aureus 6538 phage in river
By the colon bacillus in streptococcus aureus 6538 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having streptococcus aureus 6538 phage occurs.
Embodiment 7
The separation of streptococcus faecium 32221 phage in river
By the colon bacillus in streptococcus faecium 32221 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having streptococcus faecium 32221 phage occurs.
Embodiment 8
The separation of Shigellae 51424 phage in river
By the colon bacillus in Shigellae 51424 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having Shigellae 51424 phage occurs.
Embodiment 9
The separation of Vibrio parahemolyticus 20025 phage in river
By the colon bacillus in Vibrio parahemolyticus 20025 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having Vibrio parahemolyticus 20025 phage occurs.
Embodiment 10
The separation of river Small Intestine enteocolitica 52301 phage
By the colon bacillus in Yersinia enterocolitica 52301 alternative embodiment 2, other step, with embodiment 2, detects in river that the plaque having Yersinia enterocolitica 52301 phage occurs.
Embodiment 11
The separation of multiple phage in river
Host Strains colon bacillus 15597, colon bacillus O157 ︰ H721530 and Salmonellas 50001 are respectively got the colon bacillus in 1mL alternate embodiment 2, other step, with embodiment 2, detects in river that the plaque having colon bacillus 15597 phage occurs, the plaque of colon bacillus O157 ︰ H721530 phage occurs, the plaque of Salmonellas 50001 phage occurs.
Experiment proves, method of the present invention, when other testing conditions is constant, only needs to change Host Strains, is just likely separated to corresponding phage.
The description of the embodiment of the present invention is illustrative instead of determinate, can list several embodiments, therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention according to institute's limited range.
Claims (2)
1. a water pnagus medius separation method, its feature comprises the steps:
(1) preparation of positive charge silica gel particle
1. in proportion, take 6-7g aluminum chloride under room temperature slowly to join in 950mL distilled water and make dissolving; Add 45mL, 2M aqueous sodium carbonate, occur milky Al (OH)
3gel, adjust ph, to 7.0-7.5, adds water and is settled to 1000mL; At room temperature leave standstill 18-24h;
2. liquid step 1. obtained is to abandon supernatant after the centrifugal 10-30min of 1100g; In precipitation, add 0.14M sodium chloride aqueous solution to the resuspended precipitation of 1000mL, leave standstill 18-24h;
3. repeating step 2. 2-4 time, in 121 DEG C of autoclaving 20min, is cooled to normal temperature;
4. getting 1200-1400g particle diameter is that 150-300 μm of silica gel joins in the liquid that 3. step obtain, and limit edged stirs, and makes it abundant mixing, is placed in 50-100 DEG C of baking box and dries, obtain positive charge silica gel particle;
(2) preparation of elutriant
Take 30g Tryptones, 15g beef powder, 15g sodium-chlor, 30-40g glycine and 20-30g sodium hydroxide adding distil water to 1L, heating for dissolving, adjustment pH is 10.2-10.5, autoclaving 20min at 121 DEG C;
(3) separation of phage:
1. in chromatography column, add appropriate sterile purified water, then add positive charge silica gel particle and constantly stir removal bubble, with 300-700mL/min speed, water sample to be measured is flow through chromatography column from the top of chromatography column by peristaltic pump;
2., after water sample to be measured all flows through chromatography column, with being equivalent to positive charge silica gel particle quality 3-4 elution doubly, collect wash-out effluent liquid, adjustment pH is 7.0-7.5; Add PEG6000 make mass concentration be 10%-15% and under agitation make PEG6000 dissolve, leave standstill 20-40min at normal temperatures, the centrifugal 20-40min of 12000-16000g, abandon supernatant liquor, be settled to 10-100mL obtain liquid 1 by the resuspended precipitation of the PBS of 0.1MpH=7.0-7.5, in liquid 1, add 10 × liquid nutrient medium, add that volume is liquid 1 volume 1/10 of described 10 × liquid nutrient medium, obtains liquid 2;
3. separately get 1mL10 × liquid nutrient medium to add sterile saline and obtain 1 × liquid nutrient medium to 10mL, the Host Strains of phage is seeded in 1 × liquid nutrient medium, at 30-37 DEG C, cultivates 18-24h;
4. getting the bacterium liquid that 1mL step (3) 3. obtains is seeded in liquid 2,18-24h is cultivated at 30-37 DEG C, then the centrifugal 10-20min of 3000-4000g, collect supernatant liquor, whether get after bacterium liquid that 1mL supernatant liquor and 100 μ L steps (3) 3. obtain mixes and leave standstill 5min, adopting double-layer agar technique to detect in supernatant liquor has plaque to occur.
2. a kind of water pnagus medius separation method according to claim 1, is characterized in that the Host Strains of described phage is colon bacillus, Salmonellas, colon bacillus O157 ︰ H7, streptococcus aureus, streptococcus faecium, Shigellae, Vibrio parahemolyticus or Yersinia enterocolitica.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282127A (en) * | 2015-06-09 | 2017-01-04 | 菲吉乐科(南京)生物科技有限公司 | New phage, a combination thereof thing and their preparation method and application |
| CN106811457A (en) * | 2017-02-24 | 2017-06-09 | 集美大学 | One kind separates bacteriophage carrier and its preparation method and application |
| CN108699532A (en) * | 2015-12-21 | 2018-10-23 | 尹特荣生物科技株式会社 | Novel vibrio parahaemolyticus phage Vib-PAP-2 and its for inhibit vibrio parahaemolytious be proliferated purposes |
| CN113173576A (en) * | 2021-05-07 | 2021-07-27 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Graphene aerogel, preparation method and application thereof, and elution method of food-borne pathogenic microorganisms on graphene aerogel |
-
2014
- 2014-10-29 CN CN201410593832.9A patent/CN104312984B/en active Active
Non-Patent Citations (1)
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| MIN JIN 等: "Development of a Novel Filter Cartridge System with Electropositive Granule Media to Concentrate Viruses from Large Volumes of Natural Surface Water.", 《ENVIRONMENTAL SCIENCE&TECHNOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282127A (en) * | 2015-06-09 | 2017-01-04 | 菲吉乐科(南京)生物科技有限公司 | New phage, a combination thereof thing and their preparation method and application |
| CN106282127B (en) * | 2015-06-09 | 2019-07-05 | 菲吉乐科(南京)生物科技有限公司 | New bacteriophage, its composition and their preparation method and application |
| CN108699532A (en) * | 2015-12-21 | 2018-10-23 | 尹特荣生物科技株式会社 | Novel vibrio parahaemolyticus phage Vib-PAP-2 and its for inhibit vibrio parahaemolytious be proliferated purposes |
| CN108699532B (en) * | 2015-12-21 | 2022-08-23 | 尹特荣生物科技株式会社 | Vibrio parahaemolyticus bacteriophage Vib-PAP-2 and use thereof for inhibiting proliferation of Vibrio parahaemolyticus |
| CN106811457A (en) * | 2017-02-24 | 2017-06-09 | 集美大学 | One kind separates bacteriophage carrier and its preparation method and application |
| CN106811457B (en) * | 2017-02-24 | 2019-05-03 | 集美大学 | A kind of separation bacteriophage carrier and its preparation method and application |
| CN113173576A (en) * | 2021-05-07 | 2021-07-27 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Graphene aerogel, preparation method and application thereof, and elution method of food-borne pathogenic microorganisms on graphene aerogel |
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