CN107937623A - A kind of method and kit using strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus - Google Patents
A kind of method and kit using strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus Download PDFInfo
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Abstract
The present invention provides a kind of method and kit using strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus, belong to vibrio alginolyticus strain idenfication technical field, the described method comprises the following steps：1) strain to be checked is coated on LB agar plates, stands 15~20min, obtain tablet to be checked；2) strong vibrio alginolyticus bacteriophage is added dropwise on the tablet to be checked, obtains identification system；3) by the middle identification system obtained the 8~16h of quiescent culture at 28~35 DEG C of the step 2), if plaque occurs, then strain to be checked is vibrio alginolyticus.Method of the present invention can fast and accurately identify vibrio alginolyticus, have it is efficient, it is of low cost, the characteristics of save trouble and labor, rate of accuracy reached to 100%, preliminary screening and identification suitable for a large amount of vibrio alginolyticus.
Description
Technical field
The invention belongs to vibrio alginolyticus strain idenfication technical field, and in particular to a kind of fast with strong vibrio alginolyticus bacteriophage
The method and kit of speed identification vibrio alginolyticus.
Background technology
Vibrio alginolyticus is a kind of halophagia Gram-negative brevibacterium, and no gemma, pod membrane, are under the jurisdiction of vibrionaceae, vibrio,
It is a kind of common Marine Pathogenic Bacteria, its quantity is occupied first of seawater class vibrios.People can be caused wound infection, food poisoning, in
The diseases such as otitis.Meanwhile it is also a kind of conditioned pathogen of the ocean water cultivated animals such as fish, shrimp, shellfish, when environmental degradation opportunity
Body immunity function declines, and vibrio alginolyticus sickly look is easily broken out.
Due to vibrio alginolyticus physiological and biochemical property and hereditary feature it is complicated various, it is relatively tired that precise Identification is carried out to it
Difficulty, and carry out the multiple steps of identification needs and longer time with traditional Physiology and biochemistry method：The base of bacterial strain is grasped first
The physiological and biochemical property such as eigen, nutrient type, aerobic-type；Classification manual is consulted on this basis, determines which bacterial strain belongs to
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 bacterial strain
It need to identify the diagnostic characteristics for kind, then further measuring kind.As qualification result is related to new taxon, then carry out including gene
Comprehensive identification including type.With taxonomic development, microorganism classification is more and more with nucleotide sequence Phylogenetic Analysis
To establish new taxon and original taxon be adjusted.Produce the problem of new：Increasingly carry out more points by having
Class unit cannot only be differentiated with phenotypic characteristic, it is necessary to could be identified with reference to genotype detection.
The quick determination method of existing frequently-used vibrio alginolyticus 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 vibrio alginolyticus
Identification method, cultivated animals disease be monitored and prevents just to seem 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 with strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus
Method and kit.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：One kind utilizes strong vibrio alginolyticus phagocytosis
The method of body Rapid identification vibrio alginolyticus, comprises the following steps：
1) strain to be checked is coated on LB agar plates, stands 15~20min, obtain tablet to be checked；
2) strong vibrio alginolyticus bacteriophage is added dropwise on the tablet to be checked, obtains identification system；
3) by the identification system obtained in the step 2) at 28~35 DEG C 8~16h of quiescent culture, if plaque
Occur, then strain to be checked is vibrio alginolyticus.
Preferably, strong vibrio alginolyticus bacteriophage described in step 2) is to contain more than 10 kinds strong vibrio alginolyticus bacteriophages
Mixed liquor.
Preferably, the concentration of the step 1) strain to be checked is 107~109Cfu/mL, coating volume are 100~200 μ l.
Preferably, the potency of the strong vibrio alginolyticus bacteriophage described in step 2) is 108~1010Pfu/mL, is added dropwise body
Product is 20~50 μ l.
Preferably, the acquisition methods of the vibrio alginolyticus bacteriophage comprise the following steps：
The vibrio alginolyticus bacteriophage being separated to and host strain vibrio alginolyticus, be mixed by S1, obtains nutrient solution；
S2, by the nutrient solution separation of solid and liquid, collection liquid phase component is vibrio alginolyticus bacteriophage.
It is further preferred that the method for the separation of solid and liquid is centrifugation.
Further, the rotating speed of the centrifugation is 1000~12000rpm, and the time of the centrifugation is 5~20min.
Present invention also offers a kind of kit of Rapid identification vibrio alginolyticus, the kit includes strong vibrio alginolyticus
Bacteriophage.
Preferably, the potency of the strong vibrio alginolyticus bacteriophage is 108~1010pfu/mL。
It is further preferred that the kit further includes LB agar plates.
Beneficial effects of the present invention：
The identification method of vibrio alginolyticus of the present invention, infects the specificity of host strain using bacteriophage and cracks original
Reason, identifies vibrio alginolyticus using strong vibrio alginolyticus bacteriophage, can fast and accurately identify vibrio alginolyticus, have efficiency
Height, it is of low cost, the characteristics of save trouble and labor, rate of accuracy reached to 100%.
Kit of the invention using including strong vibrio alginolyticus bacteriophage as identification vibrio alginolyticus, it is of low cost, can
Quickly and accurately vibrio alginolyticus is identified, easy to use, preliminary screening and identification suitable for a large amount of vibrio alginolyticus.
Brief description of the drawings
Fig. 1 is the photo of 1 vibrio alginolyticus identification system plaque of embodiment.
Embodiment
The present invention provides a kind of method with strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus, including following step
Suddenly：
1) strain to be checked is coated on LB agar plates, stands 15~20min, obtain tablet to be checked；
2) strong vibrio alginolyticus bacteriophage is added dropwise on the tablet to be checked, obtains identification system；
3) by the identification system obtained in the step 2) at 28~35 DEG C 8~16h of quiescent culture, if plaque
Occur, then strain to be checked is vibrio alginolyticus.
The present invention does not limit the source of the strain to be checked, in specific implementation process of the present invention, the bacterium to be checked
Kind can be separated from environmental sample, such as water sample, soil sample or animal tissue etc..The strain to be checked preferably isolates and purifies
Pure bacterium afterwards, more preferably vibrios.The bacterium to be checked is preferably cultivated before identification, the incubation time of the bacterium to be checked
Preferably 8~12h；The method and condition of the culture using this area routine bacterial screening during cultural method with
Condition.The concentration of the bacterium to be checked is preferably 107~109Cfu/mL, more preferably 108cfu/mL.In the present invention,
Bacterium to be checked is coated on LB agar plates, the coating preferably carries out in gnotobasis, specifically can be in sterile working
Carried out in platform.In the present invention, the coating volume of the strain to be checked is preferably 100~200 μ l, and more preferably 120~180
μ l, are further 150 μ l.After the bacterium to be checked is coated on LB agar plates, stand.The time of the standing is preferably 15~
20min, more preferably 16~18min.The present invention obtains tablet to be checked after the standing.
Strong vibrio alginolyticus bacteriophage is added dropwise on tablet to be checked, is identified after tablet to be checked is obtained by the present invention
System.In the present invention, the strong vibrio alginolyticus bacteriophage preferably contains more than 10 kinds strong vibrio alginolyticus bacteriophages
Mixed liquor, the more preferably mixed liquor containing 15~20 kinds of strong vibrio alginolyticus bacteriophages；The strong vibrio alginolyticus bites
The potency of thalline is preferably 108~1010Pfu/mL, more preferably 109pfu/mL.Heretofore described vibrio alginolyticus phagocytosis
The dropwise addition volume of body is preferably 20~50 μ l, more preferably 30~40 μ l.In the present invention, the strong vibrio alginolyticus bacteriophage
It is preferably isolated from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.
In the present invention, the acquisition methods of the vibrio alginolyticus bacteriophage comprise the following steps：S1, the molten algae that will be separated to
Vibriophage obtains nutrient solution with host strain vibrio alginolyticus mixed culture；S2, by nutrient solution separation of solid and liquid, collect liquid phase component
For vibrio alginolyticus bacteriophage.
In the present invention, it can also be the bacterium that self-control isolates and purifies that the host strain vibrio alginolyticus, which can be commercially available strain,
Kind；Specifically the host strain vibrio alginolyticus described in implementation process of the present invention derives from sick the shrimp enteron aisle or liver pancreas of Penaeus Vannmei
Gland.The present invention vibrio alginolyticus bacteriophage and host strain vibrio alginolyticus mixed culture before, preferably to host strain vibrio alginolyticus into
Row culture.The host strain vibrio alginolyticus is preferably that the inoculation of solid single bacterium colony or seed liquor are inoculated with the present invention.When
When the host strain is inoculated with for solid single bacterium colony, the preferably aseptically single bacterium colony inoculation on picking conservation tablet,
Cultivation temperature is preferably 28~35 DEG C, more preferably 30~33 DEG C；The culture rotating speed is preferably 50~200rpm, more
Preferably 100~180rpm；Incubation time is preferably 14~18h, more preferably 15~17h.When the host strain is kind
During sub- liquid inoculation, preferably aseptically the seed liquor of 4 DEG C of preservations is inoculated in fluid nutrient medium and is cultivated；It is described
The inoculum concentration of seed liquor is preferably 10~20% (volumes), and more preferably 12~18%；The density of the seed liquor is preferred
For 106~108CFU/mL, more preferably 107CFU/mL；The incubation time is preferably 10~16h, and more preferably 12
~14h；It is consistent when cultivation temperature, rotating speed during the liquid inoculation culture are with solid single bacterium colony inoculated and cultured, it is no longer superfluous herein
State.The present invention is not particularly limited the culture medium of the Liquid Culture, and the vibrio alginolyticus culture medium using this area routine is
Can, it is preferably LB fluid nutrient mediums in the specific embodiment of the invention.
The present invention is not special to the vibrio alginolyticus bacteriophage and condition of culture that host strain vibrio alginolyticus is mixed
Limit, using the condition of culture of the vibrio alginolyticus of this area routine, above-mentioned vibrio alginolyticus place is used in specific implementation process
The condition of culture of main bacterium.
The method of the separation of solid and liquid preferably centrifuges in the present invention；The rotating speed of the centrifugation is preferably 1000~
12000rpm, more preferably 3000~8000rpm；The time of the centrifugation is preferably 5~20min, and more preferably 10
~15min；The temperature of the centrifugation is preferably 2~10 DEG C, more preferably 4~6 DEG C.In the present invention, the centrifugation knot
Shu Hou, is preferably filtered the liquid phase component being collected into, and the filtering is preferably filtered using 0.22 μm of filter, this hair
In bright, the purpose of the filtering is thoroughly to remove host strain, excludes interference of the host strain to qualification result.
The present invention is after identification system is obtained, 8~16h of quiescent culture, sight at 28~35 DEG C by the identification system of acquisition
Identification system is examined, if plaque occurs, strain idenfication to be checked is vibrio alginolyticus.The identification system is stood in the present invention
Cultivation temperature is preferably 30~33 DEG C；The time of the quiescent culture is preferably 10~14h, more preferably 12h.This hair
Bright to observe identification system after quiescent culture, if there is plaque appearance in identification system, strain idenfication to be checked is molten
Algae vibrios.
Present invention also offers a kind of kit of Rapid identification vibrio alginolyticus, the kit includes strong molten algae arc
Bacterium bacteriophage, the strong vibrio alginolyticus bacteriophage preferably comprise the mixed liquor of more than 10 kinds strong vibrio alginolyticus bacteriophages,
More preferably include the mixed liquor of 15~20 kinds of strong vibrio alginolyticus bacteriophages.The potency of the strong vibrio alginolyticus bacteriophage
Preferably 108~1010Pfu/mL, more preferably 109pfu/mL.In the present invention, the kit preferably further includes LB
Agar plate.The present invention is not particularly limited the preparation method of the kit of the vibrio alginolyticus, using above-mentioned vibrio alginolyticus
The acquisition methods of bacteriophage obtain vibrio alginolyticus bacteriophage, and then obtain the kit of identification vibrio alginolyticus.It is heretofore described
The shelf-life of kit is 3 months, and preservation temperature is 4 DEG C.The application method of heretofore described kit is with reference to above-mentioned with strong
The method of property vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus, then this repeats no more.
The molten algae arc of strong vibrio alginolyticus bacteriophage Rapid identification is utilized to one kind provided by the invention with reference to embodiment
The method of bacterium 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 alginolyticus bacteriophage Rapid identification vibrio alginolyticus：
Step A：Host strain isolates and purifies：Separation obtains vibrio alginolyticus from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis.
Step B：The expansion culture of host strain：5% NaCl, 121 DEG C of autoclavings are added in LB fluid nutrient mediums
20min, is cooled to room temperature, and obtains nutrient solution.Aseptically the vibrio alginolyticus single bacterium colony on picking conservation tablet is seeded to
In nutrient solution, 16h is cultivated under 30 DEG C, 150rpm rotating speeds, obtains host's bacteria culture fluid.
Step C：The separation of vibrio alginolyticus bacteriophage：Separation obtains vibrio alginolyticus phagocytosis from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds
Body.
Step D：Vibrio alginolyticus bacteriophage spreads cultivation：To the vibrio alginolyticus bacteriophage separated in host's bacteria culture fluid into
Row spreads cultivation, the condition of culture of host's bacteria culture fluid in the rapid B of the conditional synchronization that spreads cultivation.
Step E：The liquid 8000rpm at 4 DEG C that spreads cultivation of gained is centrifuged into 5min so that host strain falls to bottom, takes
Layer clarification part is vibrio alginolyticus bacteriophage mixed liquor, its potency is 108pfu/mL。
Step F：Using drop method, the concentration that 50 plants are incubated overnight is 109The various vibrios of cfu/mL, are inhaled with liquid-transfering gun
Take 100 μ l to drop in tablet center, then LB agar plates are spread evenly across with spreading rod, stand 20 minutes, the molten algae of 20 μ l is added dropwise
Vibriophage mixed liquor, keeps 30 DEG C of environment temperature and quiescent culture 12h, and observation whether there is plaque appearance；If so, demonstrate,prove
Bright is vibrio alginolyticus, and in 50 plants of vibrios, 36 plants plaque occur, are accredited as vibrio alginolyticus.Tested with reference to 16srDNA and Physiology and biochemistry
Card, rate of accuracy reached to 100%.
Embodiment 2
The present embodiment is included the following steps using the method for strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus：
Step A：Host strain isolates and purifies：Separation obtains vibrio alginolyticus from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis.
Step B：The expansion culture of host strain：2% NaCl, 121 DEG C of autoclavings are added in LB fluid nutrient mediums
20min, is cooled to room temperature, and obtains nutrient solution.The 10 of 15% nutrient solution volume is added under aseptic condition in nutrient solution6CFU/mL
Vibrio alginolyticus bacterium solution, cultivate 10h under 32 DEG C, 200rpm rotating speeds, obtain host's bacteria culture fluid.
Step C：The separation of vibrio alginolyticus bacteriophage：Separation obtains vibrio alginolyticus phagocytosis from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds
Body.
Step D：Vibrio alginolyticus bacteriophage spreads cultivation：To the vibrio alginolyticus bacteriophage separated in host's bacteria culture fluid into
Row spreads cultivation, the condition of culture of host's bacteria culture fluid in the rapid B of the conditional synchronization that spreads cultivation.
Step E：The liquid 5000rpm at 6 DEG C that spreads cultivation of gained is centrifuged into 10min so that host strain falls to bottom, upper strata
The filter filtering that 0.22 μm of fining end lease making, collects filtrate, is vibrio alginolyticus bacteriophage mixed liquor, its potency is after measured
109pfu/mL。
Step F：Using drop method, the concentration that 30 plants are incubated overnight is 108The various vibrios of cfu/mL, are inhaled with liquid-transfering gun
Take 150 μ l to drop in tablet center, then LB agar plates are spread evenly across with spreading rod, stand 15 minutes, the molten algae of 30 μ l is added dropwise
Vibriophage mixed liquor, keeps 34 DEG C of environment temperature and quiescent culture 9h, and observation whether there is plaque appearance；If so, demonstrate,prove
Bright is vibrio alginolyticus, and in 30 plants of vibrios, 22 plants plaque occur, are accredited as vibrio alginolyticus.Tested with reference to 16srDNA and Physiology and biochemistry
Card, rate of accuracy reached to 100%.
Embodiment 3
The present embodiment is included the following steps using the method for strong vibrio alginolyticus bacteriophage Rapid identification vibrio alginolyticus：
Step A：Host strain isolates and purifies：Separation obtains vibrio alginolyticus from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis.
Step B：The expansion culture of host strain：5% NaCl, 121 DEG C of autoclavings are added in LB fluid nutrient mediums
20min, is cooled to room temperature, and obtains nutrient solution.Aseptically the vibrio alginolyticus single bacterium colony on picking conservation tablet is seeded to
In nutrient solution, 18h is cultivated under 28 DEG C, 120rpm rotating speeds, obtains host's bacteria culture fluid.
Step C：The separation of vibrio alginolyticus bacteriophage：Separation obtains vibrio alginolyticus phagocytosis from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds
Body.
Step D：Vibrio alginolyticus bacteriophage spreads cultivation：To the vibrio alginolyticus bacteriophage separated in host's bacteria culture fluid into
Row spreads cultivation, the condition of culture of host's bacteria culture fluid in the rapid B of the conditional synchronization that spreads cultivation.
Step E：By the liquid that spreads cultivation of gained, 4000rpm centrifuges 20min at 5 DEG C so that host strain falls to bottom, takes
Layer clarification part is vibrio alginolyticus bacteriophage mixed liquor, its potency is 1010pfu/mL。
Step F：Using drop method, the concentration that 25 plants are incubated overnight is 107The various vibrios of cfu/mL, are inhaled with liquid-transfering gun
Take 200 μ l to drop in tablet center, then LB agar plates are spread evenly across with spreading rod, stand 20 minutes, the molten algae of 40 μ l is added dropwise
Vibriophage mixed liquor, keeps 28 DEG C of environment temperature and quiescent culture 16h, and observation whether there is plaque appearance；If so, demonstrate,prove
Bright is vibrio alginolyticus, and in 25 plants of vibrios, 24 plants plaque occur, are accredited as vibrio alginolyticus.Tested with reference to 16srDNA and Physiology and biochemistry
Card, rate of accuracy reached to 100%.
Embodiment 4
A kind of kit of Rapid identification vibrio alginolyticus, including the strong vibrio alginolyticus bacteriophage that embodiment 1 is prepared
Mixed liquor and LB agar plates.The shelf-life of the kit is 3 months, and preservation temperature is 4 DEG C.
The application method of the kit：
Using drop method, the concentration that 10 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 alginolyticus bacteriophage mixed liquor in μ l kits, keeps 32 DEG C of environment temperature and quiescent culture 12h, and observation whether there is plaque
Occur；If so, it is proved to be vibrio alginolyticus.Identify in 10 plants of vibrios, 7 kinds plaque occur, are accredited as vibrio alginolyticus, accurately
Rate reaches 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 alginolyticus bacteriophage Rapid identification vibrio alginolyticus, comprises the following steps：
1) strain to be checked is coated on LB agar plates, stands 15~20min, obtain tablet to be checked；
2) strong vibrio alginolyticus bacteriophage is added dropwise on the tablet to be checked, obtains identification system；
3) by the identification system obtained in the step 2) at 28~35 DEG C 8~16h of quiescent culture, if plaque goes out
Existing, then strain to be checked is vibrio alginolyticus.
2. according to the method described in claim 1, it is characterized in that, strong vibrio alginolyticus bacteriophage described in step 2) be containing
The mixed liquor of more than 10 kinds strong vibrio alginolyticus bacteriophages.
3. according to the method described in claim 1, it is characterized in that, the concentration of the step 1) strain to be checked is 107~
109Cfu/mL, coating volume are 100~200 μ l.
4. method according to claim 1 or 2, it is characterised in that the strong vibrio alginolyticus bacteriophage described in step 2)
Potency be 108~1010Pfu/mL, dropwise addition volume are 20~50 μ l.
5. according to the method described in claim 1, it is characterized in that, the acquisition methods of the vibrio alginolyticus bacteriophage are including following
Step：
The vibrio alginolyticus bacteriophage being separated to and host strain vibrio alginolyticus, be mixed by S1, obtains nutrient solution；
S2, by the nutrient solution separation of solid and liquid, collection liquid phase component is vibrio alginolyticus 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.
It is 7. described according to the method described in claim 6, it is characterized in that, the rotating speed of the centrifugation is 1000~12000rpm
The time of centrifugation is 5~20min.
8. a kind of kit of Rapid identification vibrio alginolyticus, it is characterised in that including strong vibrio alginolyticus bacteriophage.
9. kit according to claim 8, it is characterised in that the potency of the strong vibrio alginolyticus bacteriophage is 108~
1010pfu/mL。
10. kit according to claim 8 or claim 9, it is characterised in that the kit further includes LB agar plates.
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| CN110129280A (en) * | 2019-05-14 | 2019-08-16 | 菲吉乐科(南京)生物科技有限公司 | Wide fragmentation pattern vibrio alginolyticus bacteriophage and combinations thereof, kit and application |
| CN110129280B (en) * | 2019-05-14 | 2020-11-20 | 菲吉乐科(南京)生物科技有限公司 | Wide-cracking spectrum vibrio alginolyticus bacteriophage and composition, kit and application thereof |
| CN112391354A (en) * | 2019-08-14 | 2021-02-23 | 宁波大学 | Vibrio alginolyticus efficient lytic phage vB _ ValM-Yong3 and application thereof |
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