CN107988312A - A kind of method and kit using strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi - Google Patents
A kind of method and kit using strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi Download PDFInfo
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- CN107988312A CN107988312A CN201711207317.2A CN201711207317A CN107988312A CN 107988312 A CN107988312 A CN 107988312A CN 201711207317 A CN201711207317 A CN 201711207317A CN 107988312 A CN107988312 A CN 107988312A
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Abstract
The present invention provides a kind of method and kit using strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi, belong to Vibrio harveyi strain idenfication technical field, the described method comprises the following steps：1) it is 10 by concentration7~109Cfu/mL strains to be checked are coated on LB agar plates, are stood 15~20min, are obtained tablet to be checked；2) strong Vibrio harveyi bacteriophage mixed liquor is added dropwise on tablet to be checked, obtains identification system；3) identification system obtained in step 2) is placed in 28~35 DEG C, 8~16h of quiescent culture, observation identification system occurs if plaque, and strain idenfication to be checked is Vibrio harveyi.Method cost of the present invention is low, time saving and energy saving, and required time is only 16~18h, particularly suitable for the preliminary screening of a large amount of Vibrio harveyis.
Description
Technical field
The invention belongs to Vibrio harveyi strain idenfication technical field, and in particular to a kind of with strong Vibrio harveyi phagocytosis
The method and kit of body Rapid identification Vibrio harveyi.
Background technology
Vibrio harveyi belongs to Gram-negative bacteria, rod-short, both ends blunt circle, the extremely raw single flagellum of tool one.Breathe out Vickers arc
Bacterium is the main pathogen of photism vibriosis, can infect a variety of marine vertebrates and invertebrate.Photism vibriosis
It is due to gain the name when Vibrio harveyi is in high-density growth in luminescence phenomenon, the children of its main infection seedling stage animal and animal
Body, Symptoms become opaque, motility decrease, luminous and death etc. for animal body.
Due to Vibrio harveyi physiological and biochemical property and hereditary feature it is complicated various, precise Identification comparison is carried out to it
Difficulty, and carry out the multiple steps of identification needs and longer time with traditional Physiology and biochemistry method：Bacterial strain is grasped first
The physiological and biochemical property such as essential characteristic, nutrient type, aerobic-type；Classification manual is consulted on this basis, determines which bacterial strain belongs to
One major class (group, group), carries out feature and determines.The ownership scope progressively reduced according to measurement result, primarily determines that section, belongs to.Such as bacterium
Strain need to identify the diagnostic characteristics for kind, then further measuring kind.As qualification result is related to new taxon, then carry out including base
Because of the comprehensive identification including type.
The quick determination method of existing frequently-used Vibrio harveyi mainly has molecular biology method and immune diagnostic technique；
And the above method is there are complicated, the of high cost, shortcoming such as accuracy rate is low.Therefore, simple, quick, accurate, economic Kazakhstan Vickers
The identification method of vibrios, is monitored cultivated animals disease and prevents just to seem and be even more important.
The content of the invention
In view of this, it is an object of the invention to provide a kind of Vickers is breathed out with strong Vibrio harveyi bacteriophage Rapid identification
The method and kit of vibrios.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：It is a kind of with strong Vibrio harveyi phagocytosis
The method of body Rapid identification Vibrio harveyi, comprises the following steps：1) it is 10 by concentration7~109Cfu/mL strains to be checked are coated on
On LB agar plates, 15~20min is stood, obtains tablet to be checked；2) strong Vibrio harveyi bacteriophage mixed liquor is added dropwise to
On the tablet to be checked, identification system is obtained；The strong Vibrio harveyi bacteriophage mixed liquor includes more than 10 kinds strong Kazakhstan
Vickers Vibriophage；3) identification system obtained in step 2) is placed in 28~35 DEG C, 8~16h of quiescent culture, observation identification
System occurs if plaque, and strain idenfication to be checked is Vibrio harveyi.
Preferably, strong Vibrio harveyi bacteriophage mixed liquor described in step 1) includes 15~30 kinds of strong Kazakhstan Vickers
Vibriophage.
Preferably, the coating volume of the step 1) strain to be checked is 100~200 μ l, and the strong Vibrio harveyi is bitten
It is 20~50 μ l that volume, which is added dropwise, in thalline mixed liquor.
Preferably, the potency of the strong Vibrio harveyi bacteriophage described in step 2) is 108~1010pfu/mL。
Preferably, the acquisition methods of the Vibrio harveyi bacteriophage comprise the following steps：1) the Kazakhstan Vickers that will be separated to
Vibriophage obtains nutrient solution with host strain Vibrio harveyi mixed culture；2) by nutrient solution separation of solid and liquid, liquid phase group is collected
It is divided into Vibrio harveyi bacteriophage.
Preferably, the method for the separation of solid and liquid is centrifugation.
Preferably, the rotating speed of the centrifugation is 1000~8000rpm, and the time of the centrifugation is 5~20min.
Preferably, the temperature of the centrifugation is 2~10 DEG C.
Present invention also offers a kind of kit of Rapid identification Vibrio harveyi, including strong Vibrio harveyi bacteriophage
Mixed liquor, the strong Vibrio harveyi bacteriophage mixed liquor include more than 10 kinds strong Vibrio harveyi bacteriophages.
Preferably, the kit further includes LB agar plates.
Beneficial effects of the present invention：The identification method of Vibrio harveyi of the present invention, using bacteriophage to host strain
Specificity infect and cracking principle, identify Vibrio harveyi using strong Vibrio harveyi bacteriophage, cost is low, time saving province
Power, required time are only 16~18h, and can fast and accurately identify Vibrio harveyi, and Vickers arc is breathed out particularly suitable for a large amount of
The preliminary screening of bacterium.
Brief description of the drawings
Fig. 1 is the photo of 1 Vibrio harveyi identification system plaque of embodiment.
Embodiment
The present invention provides a kind of method with strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi, including with
Lower step：1) it is 10 by concentration7~109Cfu/mL strains to be checked are coated on LB agar plates, are stood 15~20min, are treated
Examine tablet；2) strong Vibrio harveyi bacteriophage mixed liquor is added dropwise on tablet to be checked, obtains identification system；The strong Kazakhstan
Vickers Vibriophage mixed liquor includes more than 10 kinds strong Vibrio harveyi bacteriophages；3) the identification body that will be obtained in step 2)
System is placed in 28~35 DEG C, 8~16h of quiescent culture, and observation identification system occurs if plaque, and strain idenfication to be checked is tieed up to breathe out
Family name vibrios.
In the present invention, it is 10 by concentration7~109Cfu/mL strains to be checked are coated on LB agar plates, and standing 15~
20min, obtains tablet to be checked.The present invention does not limit the source of the strain to be checked, in specific implementation process of the present invention,
The strain to be checked can be separated from environmental sample, such as water sample, soil sample or animal tissue etc..The strain to be checked is preferable
For the pure bacterium after isolating and purifying, more preferably vibrios.The bacterium to be checked is preferably cultivated before identification, the bacterium to be checked
Incubation time be preferably 8~12h；During the method and condition of the culture are using the bacterial screening of this area routine
Cultural method and condition.The concentration of the bacterium to be checked is preferably 108cfu/mL.In the present invention, it is preferred to will be to be checked
Bacterium is coated on LB agar plates, and in the present invention, the coating preferably carries out in gnotobasis, specifically can be sterile
Carried out in operation console；In the present invention, the coating volume of the strain to be checked is 100~200 μ l, preferably 120~180 μ
L, more preferably 150 μ l.After the bacterium to be checked is coated on LB agar plates, 15~20min is stood, the time of the standing is excellent
Choosing for 16~18min.The present invention obtains tablet to be checked after the standing.
Strong 20~50 μ l of Vibrio harveyi bacteriophage mixed liquor are added dropwise to be checked by the present invention after tablet to be checked is obtained
On tablet, identification system is obtained.The volume of the Vibrio harveyi bacteriophage mixed liquor is preferably 30~40 μ l.In the present invention
In, the strong Vibrio harveyi bacteriophage mixed liquor preferably includes more than 10 kinds strong Vibrio harveyi bacteriophages, more excellent
Choosing includes 15~20 kinds；The potency of the strong Vibrio harveyi bacteriophage mixed liquor is preferably 108~1010pfu/
ML, more preferably 109pfu/mL；The strong Vibrio harveyi bacteriophage is preferably isolated from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.
In the present invention, the acquisition methods of the Vibrio harveyi bacteriophage comprise the following steps：A) the Kazakhstan that will be separated to
Vickers Vibriophage obtains nutrient solution with host strain Vibrio harveyi mixed culture；B) by nutrient solution separation of solid and liquid, collection liquid
Phase component is Vibrio harveyi bacteriophage.
In the present invention, the host strain Vibrio harveyi, which can be commercially available strain and can also make by oneself, isolates and purifies
Strain；Specifically the host strain Vibrio harveyi described in implementation process of the present invention derives from the sick shrimp enteron aisle of Penaeus Vannmei.
The present invention is before Vibrio harveyi bacteriophage and host strain Vibrio harveyi mixed culture, preferably to host strain Vibrio harveyi
Cultivated, the culture of the host strain Vibrio harveyi in the present invention is inoculated with for solid single bacterium colony or liquid inoculation training
Support.When the host strain is inoculated with for solid single bacterium colony, the preferable aseptically single bacterium colony on picking conservation tablet
Inoculation, cultivation temperature are preferably 28~35 DEG C, more preferably 30~33 DEG C；The culture rotating speed is preferably 50~
200rpm, more preferably 100~180rpm；14~18h of incubation time.It is excellent when the host strain is liquid inoculation culture
Choosing aseptically the liquid bacterium solution of preservation is inoculated in fluid nutrient medium is cultivated；The inoculation of the liquid bacterium solution
Amount is preferably 10~20% (volumes), more preferably 12~18%；The density of the liquid bacterium solution is preferably 106~
108CFU/mL, more preferably 107CFU/mL；The incubation time is preferably 10~16h, more preferably 12~14h；Institute
Consistent when cultivation temperature, rotating speed when stating liquid inoculation culture are with solid single bacterium colony inoculated and cultured, details are not described herein.The present invention
The culture medium of the Liquid Culture is not particularly limited, using the Vibrio harveyi culture medium of this area routine.
The condition of culture that the present invention is mixed the Vibrio harveyi bacteriophage with host strain Vibrio harveyi does not have
Particular determination, using the condition of culture of the Vibrio harveyi of this area routine, is tieed up in specific implementation process using above-mentioned Kazakhstan
The condition of culture of family name's vibrios host strain.
The method of the separation of solid and liquid preferably centrifuges in the present invention；The rotating speed of the centrifugation is preferably 1000~
8000rpm, more preferably 3000~6000rpm；The time of the centrifugation is preferably 5~20min, more preferably 10~
15min；The temperature of the centrifugation is preferably 2~10 DEG C, more preferably 4~6 DEG C.
The present invention is placed in 28~35 DEG C, 8~16h of quiescent culture after identification system is obtained, by the identification system of acquisition, sees
Identification system is examined, if plaque occurs, strain idenfication to be checked is Vibrio harveyi.The identification system is quiet in the present invention
Put cultivation temperature and be preferably 30~33 DEG C；The time of the quiescent culture is preferably 10~14h, more preferably 12h.This
Invention observes identification system, if there is plaque appearance in identification system, strain idenfication to be checked is after quiescent culture
Vibrio harveyi.
Present invention also offers a kind of kit of Rapid identification Vibrio harveyi, including strong Vibrio harveyi bacteriophage
Mixed liquor, the strong Vibrio harveyi bacteriophage mixed liquor include more than 10 kinds strong Vibrio harveyi bacteriophages.It is preferred that
, the kit further includes LB agar plates.The strong Vibrio harveyi bacteriophage mixed liquor preferably includes 15~20
Kind；The potency of the strong Vibrio harveyi bacteriophage mixed liquor is preferably 108~1010Pfu/mL, more preferably
109pfu/mL.The application method of heretofore described kit is with reference to above-mentioned with strong Vibrio harveyi bacteriophage Rapid identification
The method of Vibrio harveyi, details are not described herein.
A kind of breathed out using strong Vibrio harveyi bacteriophage Rapid identification provided by the invention is tieed up with reference to embodiment
The method of family name vibrios is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The present embodiment is included the following steps using the method for strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi：
Step A：Host strain isolates and purifies, and Vibrio harveyi of the invention is separated from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of host strain, adds 5% NaCl, 121 DEG C of autoclaving 20min in LB fluid nutrient mediums, cold
But to room temperature.Single bacterium colony is inoculated with, and aseptically the single bacterium colony inoculation on picking conservation tablet, cultivation temperature are 30 DEG C, are turned
Speed is 150rpm, incubation time 16h；Step C：Bacteriophage separates, and separates and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.Step D：Bite
Thalline spreads cultivation, and spreads cultivation to the bacteriophage separated；Step E：By the nutrient solution of gained, 8000rpm is centrifuged at 4 DEG C
5min so that host strain falls to bottom, takes upper strata to clarify part, its potency is 108pfu/mL.Using drop method, by 50 plants
The concentration being incubated overnight is 107The various vibrios of cfu/mL, draw 100 μ l with liquid-transfering gun and drop in tablet center, then use spreading rod
LB agar plates are spread evenly across, stand 20 minutes, the Vibrio harveyi bacteriophage mixed liquor of 20 μ l is added dropwise, keep environment temperature
30 DEG C and quiescent culture 12h, observation whether there is plaque appearance；If so, it is proved to be Vibrio harveyi, and in 50 plants of vibrios, 36
There is plaque in strain, is accredited as Vibrio harveyi, rate of accuracy reached to 100%.
Embodiment 2
The present embodiment is included the following steps using the method for strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi：
Step A：Host strain isolates and purifies, and Vibrio harveyi of the invention is separated from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of host strain, adds 2% NaCl, 121 DEG C of autoclaving 20min in LB fluid nutrient mediums, cold
But to room temperature.Liquid bacterium solution is inoculated with, in aseptic condition, add culture volume 15% for 106The Vibrio harveyi of CFU/mL
It is 32 DEG C, rotating speed 200rpm, incubation time 10h that bacterium solution, which carries out culture cultivation temperature into LB fluid nutrient mediums,；Step C：Bite
Thalline separates, and separates and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.Step D：Bacteriophage spreads cultivation, and the bacteriophage separated is carried out
Spread cultivation；Step E：By the nutrient solution of gained, 6000rpm centrifuges 10min at 6 DEG C so that host strain falls to bottom, takes upper strata
Part is clarified, its potency is 109pfu/mL.Using drop method, the concentration that 30 plants are incubated overnight is 108The various arcs of cfu/mL
Bacterium, draws 150 μ l with liquid-transfering gun and drops in tablet center, then is spread evenly across LB agar plates with spreading rod, stands 15min, drop
Add the Vibrio harveyi bacteriophage mixed liquor of 20 μ l, keep 32 DEG C of environment temperature and quiescent culture 14h, observation, which whether there is plaque, to go out
It is existing；If so, being proved to be Vibrio harveyi, in 30 plants of vibrios, 22 plants there is plaque, are accredited as Vibrio harveyi, accurately
Rate reaches 100%.
Embodiment 3
The present embodiment is included the following steps using the method for strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi：
Step A：Host strain isolates and purifies, and Vibrio harveyi of the invention is separated from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of host strain, adds 5% NaCl, 121 DEG C of autoclaving 20min in LB fluid nutrient mediums, cold
But to room temperature.Single bacterium colony is inoculated with, and aseptically the single bacterium colony inoculation on picking conservation tablet, cultivation temperature are 28 DEG C, are turned
Speed is 120rpm, incubation time 18h；Step C：Bacteriophage separates, and separates and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.Step D：Bite
Thalline spreads cultivation, and spreads cultivation to the bacteriophage separated；Step E：By the nutrient solution of gained, at 5 DEG C 1000rpm centrifugations
20min so that host strain falls to bottom, takes upper strata to clarify part, its potency is 1010pfu/mL.Using drop method, by 25 plants
The concentration being incubated overnight is 107The various vibrios of cfu/mL, draw 200 μ l with liquid-transfering gun and drop in tablet center, then use spreading rod
LB agar plates are spread evenly across, stand 20min, the Vibrio harveyi bacteriophage mixed liquor of 20 μ l is added dropwise, keep environment temperature
28 DEG C and quiescent culture 12h, observation whether there is plaque appearance；If so, it is proved to be Vibrio harveyi, and in 25 plants of vibrios, 24
There is plaque in kind, is accredited as Vibrio harveyi, rate of accuracy reached to 100%.
Embodiment 4
A kind of kit of Rapid identification Vibrio harveyi, including 20 kinds of strong Vibrio harveyi bacteriophage mixed liquors and LB
Agar plate.The potency of the strong Vibrio harveyi bacteriophage mixed liquor is 109pfu/mL。
The application method of the kit：
Using drop method, the concentration that 25 plants are incubated overnight is 107The various vibrios of cfu/mL, 200 μ are drawn with liquid-transfering gun
L drops in the LB agar plates center in kit, then is spread evenly across LB agar plates with spreading rod, stands 20min, is added dropwise 20
Vibrio harveyi bacteriophage mixed liquor in μ l kits, keeps 28 DEG C of environment temperature and quiescent culture 12h, and observation whether there is phagocytosis
Spot occurs；If so, it is proved to be Vibrio harveyi.Identify in 10 plants of vibrios, 7 kinds plaque occur, are accredited as and breathe out Vickers arc
Bacterium, rate of accuracy reached to 100%.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of method using strong Vibrio harveyi bacteriophage Rapid identification Vibrio harveyi, comprises the following steps：
1) it is 10 by concentration7~109Cfu/mL strains to be checked are coated on LB agar plates, stand 15~20min, are obtained to be checked
Tablet；
2) strong Vibrio harveyi bacteriophage mixed liquor is added dropwise on the tablet to be checked, obtains identification system；It is described strong
Vibrio harveyi bacteriophage mixed liquor includes more than 10 kinds strong Vibrio harveyi bacteriophages；
3) identification system obtained in the step 2) is placed in 28~35 DEG C, 8~16h of quiescent culture, observes identification system, such as
Fruit has plaque appearance, then strain idenfication to be checked is Vibrio harveyi.
2. according to the method described in claim 1, it is characterized in that, strong Vibrio harveyi bacteriophage mixing described in step 2)
Liquid includes 15~30 kinds of strong Vibrio harveyi bacteriophages.
3. according to the method described in claim 1, it is characterized in that, the coating volume of the step 1) strain to be checked for 100~
200 μ l, it is 20~50 μ l that volume, which is added dropwise, in the strong Vibrio harveyi bacteriophage mixed liquor.
4. method according to claim 1 or 2, it is characterised in that the strong Vibrio harveyi phagocytosis described in step 2)
The potency of body is 108~1010pfu/mL。
5. according to the method described in claim 1, it is characterized in that, the acquisition methods of the Vibrio harveyi bacteriophage include with
Lower step：
A) the Vibrio harveyi bacteriophage being separated to and host strain Vibrio harveyi are mixed, obtain nutrient solution；
B) by the nutrient solution separation of solid and liquid, collection liquid phase component is Vibrio harveyi bacteriophage.
6. according to the method described in claim 5, it is characterized in that, the method for the separation of solid and liquid is centrifugation.
7. according to the method described in claim 6, it is characterized in that, the rotating speed of the centrifugation is 1000~8000rpm, it is described from
The time of the heart is 5~20min.
8. the method according to claim 6 or 7, it is characterised in that the temperature of the centrifugation is 2~10 DEG C.
9. a kind of kit of Rapid identification Vibrio harveyi, it is characterised in that mixed including strong Vibrio harveyi bacteriophage
Liquid, the strong Vibrio harveyi bacteriophage mixed liquor include more than 10 kinds strong Vibrio harveyi bacteriophages.
10. kit according to claim 9, it is characterised in that the kit further includes LB agar plates.
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| CN112391355A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB _ Vhas-yong3 and application thereof |
| CN112442487A (en) * | 2019-08-14 | 2021-03-05 | 宁波大学 | Vibrio harveyi high-efficiency lytic phage vB-Vhas-yong1 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 |
| CN111676197A (en) * | 2020-07-01 | 2020-09-18 | 青岛诺安百特生物技术有限公司 | Vibrio harveyi phage, phage composition and application thereof |
| CN111705041A (en) * | 2020-07-01 | 2020-09-25 | 青岛诺安百特生物技术有限公司 | Vibrio harveyi phage vB _ KaS _ PK22, phage composition and application thereof |
| CN111676197B (en) * | 2020-07-01 | 2021-12-14 | 青岛诺安百特生物技术有限公司 | Vibrio harveyi phage, phage composition and application thereof |
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