CN104073469B - The separation of cracking aquatic pathogenic bacteria phage and screening technique - Google Patents
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Abstract
The present invention relates to a kind of separation cracking aquatic pathogenic bacteria phage and screening technique, phage entitled Φ A8 29, specific name is Aeromonas veronii phage, deposit number: CGMCC No.9152；The entitled A8 of Aeromonas veronii 29, specific name is Aeromonas veronii, deposit number: CGMCC No.9093, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: on 04 25th, 2014.The invention provides screening and the authentication method of a kind of aquatic pathogenic bacteria phage, the phage of isolated can be used in prevention and controls the infection of aquatic pathogenic bacteria, it is to avoid or reduce antibiotics use in aquaculture, and the green model of development aquaculture.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of separation cracking aquatic pathogenic bacteria phage and screening side
Method.
Background technology
China national aquaculture area 5633.21kha in 2007, cultured output 4747.5 ten thousand tons, account for complete
More than the 2/3 of world aquaculture total output, cultured output exceedes fishery output.The cultivation of annual about 1/10
Area generation disease, every year because of disease loss cultured output account for 30%, direct economic loss more than 10,000,000,000 yuan,
Wherein the sickness rate of the bacterial diseases of fishes and fatality rate are all near or above 50%.
Important aquatic pathogenic bacterium includes vibrio, Aeromonas, Plesiomonas, Flavobacterium, non-lever
Pseudomonas, Rhodopseudomonas, Pasteurella Pseudomonas, Edwardsiella, Edwardsiella tarda, yersinia
Genus, Serratieae, staphylococcus, Streptococcus, Nocardia, bacillus fusiformis genus and tuberculosis Pseudomonas etc..
The prevention of bacterial disease and control in aquaculture, main realize by adding antibiotics in feedstuff.I
State's year relating to antibiotic-producing is 400,000 tons, and wherein nearly half cultivates for animal husbandry and fishery.The knot of life-time service antibiotics
Really, the antibacterial directly resulting in 90% has drug resistance to more than one antibiotics, and the antibacterial of 20% is to more than 5 kinds
Antibiotics has drug resistance, reduces therapeutic effect, causes environmental pollution, and food safety is produced very big prestige
The side of body.Therefore, we are in the urgent need to a replacement method that can control aquatic pathogenic bacteria.
Phage (Bacteriophage or phage) is the biology that nature exists that quantity is most, there are about 1031
Individual phage.Phage is the virus of the microorganisms such as bacterial infection, it is possible to crack Host Strains specifically, simultaneously with
Whether drug resistance is unrelated for Host Strains.Phage does not infect the antibacterial of the mankind, animal, plant and other nonhost,
Almost environment is not produced pressure.On the other hand, some phage can encode a kind of enzyme, and can degrade host
The extracellular polysaccharide polymer (EPS) of bacterium, thus destroy the biofilm that pathogenic bacterium are formed.These characteristics make phagocytosis
Body can be used to control the infection of pathogenic bacterium.
Although phage is widely present in the various environment of nature, but the distribution of phage exists uncertain
Property.In the case of a lot, using conventional method, be difficult to be separated to phage, this situation occurs in multiple causing a disease
The separation of bacterium phage.The method that the present invention relates to, uses and separates pathogenic bacterium from specific environment, and cause a disease with this
Bacterium is Host Strains, from same sample, is directly separated specific phage, solves deriving from of phage
Specificity issues, the phage being separated to can be directly used for the prevention and control of aquatic products bacterial disease.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that one efficiently and rapidly Aquatic product causes a disease
The screening of bacterium phage and authentication method, use this method can efficiently and rapidly screen for different pathogenic bacterium
Phage, it is possible to avoid or reduce antibiotics use in aquaculture, for developing the green of aquaculture
Pattern, for food safety, all has important value
Another one purpose of the present invention is to separate phage and Vickers gas unit cell, name and preservation.
It is an object of the invention to be achieved through the following technical solutions
One strain can crack the phage of Aeromonas veronii, and phage entitled Φ A8-29, specific name is
Aeromonas veronii phage, deposit number: CGMCC No.9152；Depositary institution: China Microbiological
Culture presevation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Preservation date: on 04 25th, 2014.
The Aeromonas veronii of phage Φ A8-29 cracking, entitled A8-29, specific name is Aeromonas
Veronii, deposit number: CGMCC No.9093, depositary institution: Chinese microorganism strain preservation management is entrusted
Member's meeting common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: 2014
04 month 25 days year.
A kind of separation cracking aquatic pathogenic bacteria phage and screening technique, step is as follows:
(1) the separation of aquatic pathogenic bacteria
Collect various aquatic products sample, by sample homogenization, dilute homogenate, take diluent 100 μ l at selectivity
Coated plate in culture medium, selective medium includes SS culture medium, TCBS culture medium and Mai Kangkai culture medium,
It is inverted for 37 DEG C and cultivates 24-48h, picking colony, purification bacterial strain of ruling on same Selective agar medium, repeatedly
Purification three times, by the toothpick picking list bacterium colony of bacterial strain sterilizing consistent for form, accesses in LB fluid medium,
37 DEG C of shaken cultivation 12h, phase bacterium solution of taking the logarithm is placed on-80 DEG C of Storage in refrigerator with glycerite mixing；
(2) the separation of phage
The each 1g of intestinal contents of all cultivation kinds is placed in 50mlTSB-YE culture medium, 37 DEG C of shakes
Swing cultivation 72h, culture is added 50ml centrifuge tube, adds 1ml chloroform and mix gently several times, 10000 × g
Centrifugal 5min, takes supernatant and is placed in 4 DEG C of storages, and this supernatant is as the source separating phage；
(3) cultivate the aquatic pathogenic bacteria of above-mentioned separation to exponential phase, mix with the supernatant of above-mentioned preparation, then
With 0.6% agar culture medium mixing double-layer plate, cultivate 4-8h for 37 DEG C, observe the appearance of plaque, choose
Take single plaque, be placed in LB fluid medium resuspended, prepare phage solution, dilute phage solution,
Mix with the indicator bacteria of logarithmic (log) phase, then mix double-layer plate with 0.6% agar culture medium, cultivate 4h for 37 DEG C
Rear observation plaque, repeats this step purified phage,
(4) the preparation of Host genomic DNA
Host Strains is inoculated in LB fluid medium, 37 DEG C of shaken cultivation overnight after, bacterium solution is centrifugal abandons supernatant,
Precipitation adds the resuspended thalline of TE buffer, adds lysozyme room temperature and place 10min, add SDS and albumen
After enzyme K mixing, 60 DEG C of water bath heat preservation 6h, add isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1),
Vibration mixing, centrifuging and taking supernatant is in new centrifuge tube, and extracting is not to having albuminous coat；In supernatant
Add chloroform, take supernatant in new centrifuge tube, in supernatant, add 95% ethanol of 2 times of volumes
(pre-cooling), vibration mixing, it is centrifuged and discards supernatant；Adding 70% ethanol in precipitation, 10000rpm is centrifuged 5
Min, discards supernatant, is dried under room temperature；Add distilled water or TE buffer (pH8.0), it is thus achieved that Host Strains
Chromosomal DNA solution, detects with 0.8% agarose gel electrophoresis,
(5) expand the 16SrRNA gene of Host Strains
Using PCR method, the 16SrRNA genetic fragment of amplification Host Strains, pcr amplification product directly enters
Row determined dna sequence, it is thus achieved that sequence, through homology analysis, determines the biological classification of indicator bacteria,
(6) phage preserves
Phage after purification, is expanded in corresponding Host Strains by phage, degerming with membrane filtration, adds nothing
Bacterium glycerol, mixing ,-80 DEG C save backup；
(7) phage titer measures
Being diluted by phage solution, phage diluent mixes with the Host Strains of exponential phase, double-layer plate,
After 37 DEG C are cultivated 4-6h, carry out plaque count, calculate phage titer or titre,
(8) the host range analysis of phage
Cultivate the antibacterial that the Host Strains of phage is close or identical with other classification, by double-layer agar technique or direct
Point sample method, observes the collection of illustrative plates of the above-mentioned antibacterial of phage splitting, determines the host range of this phage.
Advantages of the present invention and good effect are as follows:
1, the phage that the application separates is for developing the phage preparation for important aquatic pathogenic bacteria, controls
Bacterial infection by the microbial aquatic products that cause a disease, it is to avoid or reduce antibiotics making in aquaculture process
With, reduce the enrichment of multi-drug resistant antibacterial, significant to food safety.
2, the method that the present invention provides can efficiently and rapidly screen the phage for different pathogenic bacterium, it is possible to
Avoid or reduce antibiotics use in aquaculture, for developing the green model of aquaculture, for food
Product safety, all has important value.
3, the phage name Φ A8-29 of present invention screening, specific name is Aeromonas veronii phage,
Deposit number: CGMCC No.9152；Titration is 3.3 × 1010Pfu/ml, the strong phagocytosis of isolated
Physical ability is enough in prevention and controls the infection of aquatic pathogenic bacteria, it is to avoid or reduce antibiotics making in aquaculture
With, the green model of development aquaculture.
Detailed description of the invention
Being described in further detail invention below by specific embodiment, following example are illustrative, are not
Determinate, it is impossible to limit protection scope of the present invention with this.
A kind of separation cracking aquatic pathogenic bacteria phage and screening technique, step is as follows:
(1) the separation of aquatic pathogenic bacteria
Collect various aquatic products sample, by sample homogenization, dilute homogenate, take diluent 100 μ l at selectivity
Coated plate in culture medium, selective medium includes SS culture medium, TCBS culture medium and Mai Kangkai culture medium,
It is inverted for 37 DEG C and cultivates 24-48h, picking colony, purification bacterial strain of ruling on same Selective agar medium, repeatedly
Purification three times, by the toothpick picking list bacterium colony of bacterial strain sterilizing consistent for form, accesses in LB fluid medium,
37 DEG C of shaken cultivation 12h, phase bacterium solution of taking the logarithm is placed on-80 DEG C of Storage in refrigerator with glycerite mixing；
(2) the separation of phage
The each 1g of intestinal contents of all kinds (fish) is placed in 50ml TSB-YE culture medium, 37 DEG C
72h is cultivated in concussion, culture adds 50ml centrifuge tube, adds 1ml chloroform and mix gently several times, and 10000
× g is centrifuged 5min, takes supernatant and is placed in 4 DEG C of storages, and this supernatant is as the source separating phage.
(3) cultivate the aquatic pathogenic bacteria of above-mentioned separation to exponential phase, mix with the supernatant of above-mentioned preparation, then
With 0.6% agar culture medium mixing double-layer plate, cultivate 4-8h for 37 DEG C, observe the appearance of plaque.Choose
Take single plaque, be placed in LB fluid medium resuspended, prepare phage solution, dilute phage solution,
Mix with the indicator bacteria of logarithmic (log) phase, then mix double-layer plate with 0.6% agar culture medium, cultivate 4h for 37 DEG C
Rear observation plaque, repeats this step purified phage.
(4) the preparation of Host genomic DNA
Host Strains is inoculated in LB fluid medium, 37 DEG C of shaken cultivation overnight after, bacterium solution is centrifugal abandons supernatant,
Precipitation adds the resuspended thalline of TE buffer, adds lysozyme room temperature and place 10min, add SDS and albumen
After enzyme K mixing, 60 DEG C of water bath heat preservation 6h, add isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1),
Vibration mixing, centrifuging and taking supernatant is in new centrifuge tube, and extracting is not to having albuminous coat；In supernatant
Add chloroform, take supernatant in new centrifuge tube, in supernatant, add 95% ethanol of 2 times of volumes
(pre-cooling), vibration mixing, it is centrifuged and discards supernatant；Adding 70% ethanol in precipitation, 10000rpm is centrifuged 5
Min, discards supernatant, is dried under room temperature；Add distilled water or TE buffer (pH8.0), it is thus achieved that Host Strains
Chromosomal DNA solution, with 0.8% agarose gel electrophoresis detection.
(5) expand the 16S rRNA gene of Host Strains
Using PCR method, the 16S rRNA genetic fragment of amplification Host Strains, pcr amplification product directly enters
Row determined dna sequence, it is thus achieved that sequence, through homology analysis, determines the biological classification of indicator bacteria.
(6) phage preserves
Phage after purification, is expanded in corresponding Host Strains by phage, degerming with membrane filtration, adds nothing
Bacterium glycerol, mixing ,-80 DEG C save backup；
(7) phage titer measures
Being diluted by phage solution, phage diluent mixes with the Host Strains of exponential phase, double-layer plate,
After 37 DEG C are cultivated 4-6h, carry out plaque count, calculate phage titer or titre.
(8) the host range analysis of phage
Cultivate the antibacterial that the Host Strains of phage is close or identical with other classification, by double-layer agar technique or direct
Point sample method, observes the collection of illustrative plates of the above-mentioned antibacterial of phage splitting, determines the host range of this phage.
Screening embodiment 1
According to technique scheme, it is separated to 1 strain and can crack the phage of Aeromonas veronii and corresponding place
The strain of main bacterium Aeromonas veronii 1.Phage entitled Φ A8-29, specific name is Aeromonas veronii
Phage, deposit number: CGMCC No.9152；The entitled A8-29 of Aeromonas veronii, specific name is
Aeromonas veronii, deposit number: CGMCC No.9093, depositary institution: Chinese microorganism strain
Preservation administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation
Date: on 04 25th, 2014.
(1) specimen: the aquatic products samples such as the fresh-water fishes of collection different cultivars.
(2) sample disposal: collect the gastrointestinal content of fish, be placed in 1.5ml centrifuge tube, 4 DEG C of preservations.
(3) the separation of aquatic pathogenic bacteria: being mixed by gastrointestinal content, suspension normal saline does serial dilution.
Take 10 respectively-1With 10-2Dilution liquid 100 μ l, at Selective solid culture medium SS, TCBS and Mai Kang
Triumphant upper coated plate respectively, is coated with the spreader of sterilizing, is applied to uniformly, wait flat board to be dried rear 37 DEG C of inversions
Cultivate 24-48h, in incubator, take out flat board, picking colony, selective medium carries out three zonings
Line, is inverted for 37 DEG C and cultivates 12-16h, repeatedly purification three times, chosen by the toothpick of bacterial strain sterilizing consistent for form
Take single bacterium colony, access in 5mlLB fluid medium, 37 DEG C of 200rpm shaken cultivation 12h, take the logarithm
Phase bacterium solution 1ml adds cryopreservation tube, protects in-80 DEG C of refrigerators after being subsequently added into the glycerite mixing of 0.5ml50%
Hide.
(4) the enrichment of phage: take each 1ml of intestinal contents, is placed in 50mlTSB-YE culture medium, 37 DEG C
72h is cultivated in concussion.Culture is added 50ml centrifuge tube, adds after 1ml chloroform mixes several times gently, 10000
× g is centrifuged 5min, takes supernatant and is placed in 4 DEG C of storages.Now, mixing supernatant is as separating phage
Source.
(5) the isolation and purification of phage: take 100 μ l supernatant in centrifuge tube, add 200 μ l and be in logarithm
The indicator bacteria mixing of trophophase, after 10min, by the semi-solid LB culture medium (60 of mixture Yu 5mL0.6%
DEG C) mixing, double-layer plate, cultivate 4h for 37 DEG C, observe the appearance of plaque.Bite with inoculating loop picking
Bacterial plaque, is resuspended in phage in 1mLLB fluid medium.Dilution phage solution, takes each dilute respectively
Degree of releasing 100 μ l, respectively with 200 μ l host bacteria suspension mixing 5min, then with 5mL0.6% agar culture medium
(60 DEG C) mix, and pour into rapidly in 1.5% bottom-layer agar culture medium, and after 37 DEG C are cultivated 4h, observation is bitten
The form of bacterial plaque, repeats this step purification plaque.
(6) the preservation of phage: picking plaque, mixes with the Host Strains of logarithmic (log) phase, cultivates 4h, adds for 37 DEG C
Enter several chloroforms, place 10min, 10000 × g and be centrifuged 5min, take supernatant 0.22 μm membrane filtration
The most degerming, add sterile glycerol so that it is final concentration of 30% (v/v), mixing is placed on-80 DEG C of preservations
Standby.
(7) the Molecular Identification of Host Strains: first prepare the genomic DNA of Host Strains, picking has separated phagocytosis
Single bacterium colony of the Host Strains of body, is inoculated in 5mLLB fluid medium, 37 DEG C of shaken cultivation 8-10h；
Taking bacterium solution 1.5mL after enrichment culture, 10000rpm is centrifuged 4-5min；Abandon supernatant, precipitation adds 0.5
The resuspended thalline of mL TE buffer；Adding 20mg/mL lysozyme 10 μ l, room temperature places 10min；Add
20%SDS10 μ l, shakes up, and adds 10mg/mL E.C. 3.4.21.64 50 μ l, vibration mixing, and 60 DEG C of water-baths are protected
Temperature 2h；Add isopyknic phenol: chloroform: isoamyl alcohol (25: 24: 1), vibration mixes, 10000rpm
Centrifugal 10min, takes supernatant in new centrifuge tube, repeats phenol chloroform extraction 3-7 time；In supernatant
Adding equal-volume chloroform, vibration mixing, 10000rpm is centrifuged 10min, takes supernatant and is centrifuged in new
Guan Zhong；Adding the NaAc (3mol/L) of 1/10 volume in supernatant, 95% ethanol of 2 times of volumes is (pre-
Cold), vibration mixing, 10000rpm is centrifuged 10min, discards supernatant；Precipitation adds 200 μ l70%
Ethanol, 10000rpm is centrifuged 5min, discards ethanol, is dried under room temperature；Add 100-200 μ L distilled water
Or TE buffer (pH8.0) resuspended precipitation；0.8% agarose gel electrophoresis detection.
Utilize PCR method, using the DNA of preparation as template, expand 16SrDNA genetic fragment, used
Primer sequence includes forward primer: 5 ＇-AGAGTTTGATCCTGGCTCAG-3 ＇ (E.coli bases 8 to
27) and reverse primer: 5 ＇-TACCTTGTTACGACTT-3 ＇ (E.coli bases 1507to1492).Expand
Volume increase thing direct Sequencing, it is thus achieved that sequence in GenBank, utilize Basic Local Alignment Search
Tool (BLAST 2.2.26) instrument carries out homology analysis comparison, determines that Host Strains is Aeromonas veronii.
(8) phage titer measures: phage solution normal saline does serial dilution, takes 10 respectively-6、10-7
With 10-8The each 100 μ l of diluent, the Aeromonas with 200 μ l exponential phases is mixed homogeneously respectively, falls double
Layer flat board, after 37 DEG C are cultivated 4-6h, records plaque quantity, calculates phage titer, and phage solution drips
Degree or titer are 3.3 × 1010pfu/ml。
Screening embodiment 2
Take identical technical scheme and step, gather different aquatic products specimen, isolated temperature from specimen
It is 3.0 × 10 with the phage Ф A33 of Aeromonas, the titer of this phage or titre9pfu/ml。
Screening embodiment 3
Taking identical technical scheme and step, gather different aquatic products specimen, from specimen, isolated is general
The phage Ф P5-9 of logical Bacillus proteus, the titer of this phage or titre are 6.5 × 106pfu/ml。
During aquaculture, it is possible to cause the important aquatic pathogenic bacterium of bacterial disease to have multiple, including
Vibrio, Aeromonas, Plesiomonas, Flavobacterium, acinetobacter, Rhodopseudomonas, Pasteur
Bacillus Pseudomonas, Edwardsiella, Edwardsiella tarda, Yersinia, Serratieae, staphylococcus
Genus, Streptococcus, Nocardia, bacillus fusiformis genus and tuberculosis Pseudomonas etc..The method utilizing the present invention, can
Efficiently and rapidly to screen the phage for different pathogenic bacterium, it is possible to avoid or reduce antibiotics at aquaculture
In use, for develop aquaculture green model, for food safety, all there is important value.
Claims (1)
1. a strain can crack the phage of Aeromonas veronii, it is characterised in that: phage is entitled
Φ A8-29, specific name is Aeromonas veroniiphage, deposit number: CGMCC No.9152；Protect
Tibetan unit: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Chaoyang District, Beijing City
North Star West Road 1 institute 3, preservation date: on 05 06th, 2014.
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| CN106282298A (en) * | 2015-05-13 | 2017-01-04 | 北京良润生物科技有限公司 | A kind of preparation method of selective medium |
| CN105648109B (en) * | 2016-01-08 | 2019-04-09 | 河南医学高等专科学校 | Chemistry antibacterial agent screening technique when a kind of bacteriophage sterilizes |
| CN106011081A (en) * | 2016-06-14 | 2016-10-12 | 苏州埃瑞特生物技术有限公司 | Large-scale production method of bacteriophage |
| CN106085967A (en) * | 2016-06-14 | 2016-11-09 | 苏州埃瑞特生物技术有限公司 | A kind of production method of high-titer phage |
| CN107686832B (en) * | 2016-08-05 | 2020-08-14 | 菲吉乐科(南京)生物科技有限公司 | Novel vibrio parahaemolyticus bacteriophage, and composition, preparation method and application thereof |
| CN109517803A (en) * | 2018-04-26 | 2019-03-26 | 天津科技大学 | A kind of bacteriophage intensifying method suitable for artemia nauplii |
| CN110093321B (en) * | 2019-04-30 | 2021-07-13 | 上海海洋大学 | Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases |
| CN110172451A (en) * | 2019-05-05 | 2019-08-27 | 昆明理工大学 | A kind of method of high-throughput isolation bacteriophage |
| CN112391357A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Aeromonas sobria efficient lytic phage vB _ AsoP-yong and application thereof |
| CN112391355B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio harveyi efficient lytic phage vB_VhaS-yong3 and application thereof |
| CN112391358B (en) * | 2019-08-14 | 2023-12-01 | 宁波大学 | Vibrio mediterranean virulent phage vB_VmeM-Yong and application thereof |
| CN110592027A (en) * | 2019-09-16 | 2019-12-20 | 青岛农业大学 | Aeromonas hydrophila phage separation method |
| CN110592028A (en) * | 2019-09-24 | 2019-12-20 | 青岛农业大学 | Acinetobacter johnsonii phage separation method |
| CN116478256B (en) * | 2021-01-19 | 2024-05-14 | 西南大学 | Bacteriocin produced by lactobacillus fermentum and application thereof |
| CN114395537A (en) * | 2022-01-17 | 2022-04-26 | 青岛润达生物科技有限公司 | Phage preparation and method for cleaning bottom mud of aquaculture site |
| CN116676277B (en) * | 2023-07-28 | 2023-11-03 | 中国海洋大学 | Deep-brillouin salt monad lysogenic phage and separation method and application thereof |
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