CN107828853A - A kind of method and kit using strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious - Google Patents
A kind of method and kit using strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious Download PDFInfo
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- CN107828853A CN107828853A CN201711297834.3A CN201711297834A CN107828853A CN 107828853 A CN107828853 A CN 107828853A CN 201711297834 A CN201711297834 A CN 201711297834A CN 107828853 A CN107828853 A CN 107828853A
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Abstract
The invention provides a kind of method and kit using strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious, belong to vibrio parahaemolytious 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 flat board to be checked；2) strong vibrio parahaemolyticus phage mixed liquor is added dropwise on flat board 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 parahaemolytious.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 parahaemolytious.
Description
Technical field
The invention belongs to vibrio parahaemolytious strain idenfication technical field, and in particular to a kind of with strong vibrio parahaemolytious phagocytosis
The method and kit of body Rapid identification vibrio parahaemolytious.
Background technology
Vibrio parahaemolytious belong to be gram-negative bacteria stick shape, arcuation, oval shape without Bacillus, belong to vibrio, be
A kind of common pathogen, is widely present in seawater and marine product, is the common food poisoning pathogen in China's Coastal Areas.
Due to vibrio parahaemolytious 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), carry out feature determination.The ownership scope progressively reduced according to measurement result, primarily determine that section, category.Such as bacterium
Strain need to identify the diagnostic characteristics for kind, then further determining 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 parahaemolytious mainly has molecular biology method and immune diagnostic technique；
And there is complex operation, the shortcomings such as cost is high, accuracy rate is low in the above method.Therefore, simple, quick, accurate, economic secondary haemolysis
The authentication method of vibrios, is monitored to 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 with strong vibrio parahaemolyticus phage Rapid identification pair haemolysis
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 parahaemolytious phagocytosis
The method of body Rapid identification vibrio parahaemolytious, 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 flat board to be checked；2) strong vibrio parahaemolyticus phage mixed liquor is added dropwise to
On the flat board to be checked, identification system is obtained；The strong vibrio parahaemolyticus phage mixed liquor includes more than 10 kinds strong pairs
Hemolysis vibrion bacteriophage；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 parahaemolytious.
Preferably, strong vibrio parahaemolyticus phage mixed liquor described in step 1) includes 15~30 kinds of strong secondary haemolysis
Vibriophage.
Preferably, the coating volume of the step 1) strain to be checked is 100~200 μ l, and the strong vibrio parahaemolytious bites
It is 20~50 μ l that volume, which is added dropwise, in thalline mixed liquor.
Preferably, the potency of the strong vibrio parahaemolyticus phage described in step 2) is 108~1010pfu/mL。
Preferably, the acquisition methods of the vibrio parahaemolyticus phage comprise the following steps：1) the secondary haemolysis that will be separated to
Vibriophage obtains nutrient solution with Host Strains vibrio parahaemolytious mixed culture；2) by nutrient solution separation of solid and liquid, liquid phase group is collected
It is divided into vibrio parahaemolyticus phage.
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 parahaemolytious, including strong vibrio parahaemolyticus phage
Mixed liquor, the strong vibrio parahaemolyticus phage mixed liquor include more than 10 kinds strong vibrio parahaemolyticus phages.
Preferably, the kit also includes LB agar plates.
Beneficial effects of the present invention：The authentication method of vibrio parahaemolytious of the present invention, using bacteriophage to Host Strains
Specificity infect and cracking principle, identify vibrio parahaemolytious using strong vibrio parahaemolyticus phage, cost is low, time saving province
Power, required time are only 16~18h, and can fast and accurately identify vibrio parahaemolytious, particularly suitable for a large amount of secondary haemolysis arcs
The preliminary screening of bacterium.
Brief description of the drawings
Fig. 1 is the photo of the vibrio parahaemolytious identification system plaque of embodiment 1.
Embodiment
The invention provides a kind of method with strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious, 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 flat board；2) strong vibrio parahaemolyticus phage mixed liquor is added dropwise on flat board to be checked, obtains identification system；The strong pair
Hemolysis vibrion bacteriophage mixed liquor includes more than 10 kinds strong vibrio parahaemolyticus phages；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 secondary molten
Blood 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, obtain flat board to be checked.The present invention is not limited the source of the strain to be checked, in specific implementation process of the present invention,
The strain to be checked can separate 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 conventional bacterial screening in this area
Cultural method and condition.
The concentration of the bacterium to be checked is preferably 108cfu/mL.In the present invention, it is preferred to bacterium to be checked is coated on LB fine jades
On fat flat board, in the present invention, the coating is preferably carried out in gnotobasis, can specifically be entered in aseptic operating platform
OK；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
For 150 μ l.After the bacterium to be checked is coated on LB agar plates, 15~20min is stood, time of the standing is preferably 16~
18min.The present invention obtains flat board to be checked after the standing.
Strong vibrio parahaemolyticus phage mixed liquor is added dropwise on flat board to be checked by the present invention after flat board to be checked is obtained,
Obtain identification system.The volume of the vibrio parahaemolyticus phage mixed liquor is preferably 20~50 μ l, more preferably 30~40 μ
l.In the present invention, the strong vibrio parahaemolyticus phage mixed liquor is preferably bitten including more than 10 kinds strong vibrio parahaemolytious
Thalline, more preferably including 15~25 kinds；The potency of described strong vibrio parahaemolyticus phage mixed liquor is preferably 108~
1010Pfu/mL, more preferably 109pfu/mL；The strong vibrio parahaemolyticus phage is preferably isolated from Penaeus Vannmei
Culturing pool.
In the present invention, the acquisition methods of the vibrio parahaemolyticus phage comprise the following steps：A) the pair that will be separated to
Hemolysis vibrion bacteriophage obtains nutrient solution with Host Strains vibrio parahaemolytious mixed culture；B) by nutrient solution separation of solid and liquid, collection liquid
Phase component is vibrio parahaemolyticus phage.
In the present invention, the Host Strains vibrio parahaemolytious can be that commercially available strain can also be made by oneself and isolate and purify
Strain；Specifically the Host Strains vibrio parahaemolytious described in implementation process of the present invention derives from the sick shrimp enteron aisle of Penaeus Vannmei.
The present invention is before vibrio parahaemolyticus phage and Host Strains vibrio parahaemolytious mixed culture, preferably to Host Strains vibrio parahaemolytious
Cultivated, the culture of the Host Strains vibrio parahaemolytious in the present invention is that solid single bacterium colony is inoculated with or liquid inoculation is trained
Support.When described Host Strains are that solid single bacterium colony is inoculated with, the preferable aseptically single bacterium colony on picking conservation flat board
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 Strains are 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 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
It is consistent when cultivation temperature, rotating speed when stating liquid inoculation culture are with solid single bacterium colony inoculated and cultured, it will not be repeated here.The present invention
The culture medium of the Liquid Culture is not particularly limited, using the conventional vibrio parahaemolytious culture medium in this area.
The condition of culture that the present invention is mixed to the vibrio parahaemolyticus phage with Host Strains vibrio parahaemolytious does not have
Particular determination, it is molten using above-mentioned pair in specific implementation process using the condition of culture of the conventional vibrio parahaemolytious in this area
The condition of culture of blood vibrios Host Strains.
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 parahaemolytious.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 terminates
Vibrio parahaemolytious.
Present invention also offers a kind of kit of Rapid identification vibrio parahaemolytious, including strong vibrio parahaemolyticus phage
Mixed liquor, the strong vibrio parahaemolyticus phage mixed liquor include more than 10 kinds strong vibrio parahaemolyticus phages.It is preferred that
, the kit also includes LB agar plates.The strong vibrio parahaemolyticus phage mixed liquor preferably includes 15~20
Kind；The potency of described strong vibrio parahaemolyticus phage 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 parahaemolyticus phage Rapid identification
The method of vibrio parahaemolytious, will not be repeated here.
With reference to embodiment to provided by the invention a kind of molten using strong vibrio parahaemolyticus phage Rapid identification pair
The method of blood vibrios is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The present embodiment is comprised the following steps using the method for strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious：
Step A：Host Strains isolate and purify, and vibrio parahaemolytious of the invention separates from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of Host Strains, 5% NaCl, 121 DEG C of autoclaving 20min are added in LB fluid nutrient mediums, it is cold
But to room temperature.Single bacterium colony is inoculated with, and aseptically the single bacterium colony inoculation on picking conservation flat board, cultivation temperature are 30 DEG C, are turned
Speed is 150rpm, incubation time 16h；Step C：Virulent phage separates, and separates and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds, strong
Bacteriophage species is 18 kinds.Step D：Bacteriophage is spread cultivation, and the bacteriophage separated is spread cultivation；Step E：By the training of gained
Nutrient solution, 8000rpm centrifuges 5min at 4 DEG C so that Host Strains fall to bottom, take upper strata to clarify part, and its potency is
108pfu/mL.Using drop method, the concentration that 50 plants are incubated overnight is 107Cfu/mL various vibrios, 100 are drawn with liquid-transfering gun
μ l drop in flat board center, then are spread evenly across LB agar plates with spreading rod, stand 20 minutes, 20 μ l vibrio parahaemolytious is added dropwise
Bacteriophage mixed liquor, keeps 30 DEG C of environment temperature and quiescent culture 12h, and observation whether there is plaque appearance；If so, it is proved to be
Vibrio parahaemolytious, in 50 plants of vibrios, 32 plants there is plaque, are accredited as vibrio parahaemolytious, rate of accuracy reached to 100%.
Embodiment 2
The present embodiment is comprised the following steps using the method for strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious：
Step A：Host Strains isolate and purify, and vibrio parahaemolytious of the invention separates from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of Host Strains, 2% NaCl, 121 DEG C of autoclaving 20min are added in LB fluid nutrient mediums, it is cold
But to room temperature.Liquid bacterium solution is inoculated with, in aseptic condition, add culture volume 15% for 106CFU/mL vibrio parahaemolytious
It is 32 DEG C, rotating speed 200rpm, incubation time 10h that bacterium solution carries out cultivating cultivation temperature into LB fluid nutrient mediums；Step C：It is strong
Property bacteriophage separation, separate and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds, virulent phage species be 21 kinds.Step D：Bacteriophage is expanded
Training, spreads cultivation to the bacteriophage separated；Step E：By the nutrient solution of gained, 6000rpm centrifuges 10min at 6 DEG C, makes
Obtain Host Strains and fall to bottom, take upper strata to clarify part, its potency is 109pfu/mL.Using drop method, 30 plants are incubated overnight
Concentration be 108Cfu/mL various vibrios, draw 150 μ l with liquid-transfering gun and drop in flat board center, then with spreading rod even spread
In LB agar plates, 15min is stood, 20 μ l vibrio parahaemolyticus phage mixed liquor is added dropwise, 32 DEG C of environment temperature of holding is simultaneously quiet
Culture 14h is put, observation whether there is plaque appearance；If so, being proved to be vibrio parahaemolytious, in 45 plants of vibrios, 28 plants are bitten
Bacterial plaque, it is accredited as vibrio parahaemolytious, rate of accuracy reached to 100%.
Embodiment 3
The present embodiment is comprised the following steps using the method for strong vibrio parahaemolyticus phage Rapid identification vibrio parahaemolytious：
Step A：Host Strains isolate and purify, and vibrio parahaemolytious of the invention separates from the sick shrimp enteron aisle of a large amount of Penaeus Vannmeis and obtained
.Step B：The expansion culture of Host Strains, 5% NaCl, 121 DEG C of autoclaving 20min are added in LB fluid nutrient mediums, it is cold
But to room temperature.Single bacterium colony is inoculated with, and aseptically the single bacterium colony inoculation on picking conservation flat board, cultivation temperature are 28 DEG C, are turned
Speed is 120rpm, incubation time 18h；Step C：Virulent phage separates, and separates and obtains from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds, strong
Bacteriophage species is 15 kinds.Step D：Bacteriophage is spread cultivation, and the bacteriophage separated is spread cultivation；Step E：By the training of gained
Nutrient solution, at 5 DEG C 1000rpm centrifuge 20min so that Host Strains fall to bottom, take upper strata to clarify part, its potency is
1010pfu/mL.Using drop method, the concentration that 25 plants are incubated overnight is 107Cfu/mL various vibrios, are drawn with liquid-transfering gun
200 μ l drop in flat board center, then are spread evenly across LB agar plates with spreading rod, stand 20min, 20 μ l secondary haemolysis arc is added dropwise
Bacterium bacteriophage mixed liquor, keeps 28 DEG C of environment temperature and quiescent culture 12h, and observation whether there is plaque appearance；If so, prove
It is vibrio parahaemolytious, in 20 plants of vibrios, 16 kinds plaque occur, are accredited as vibrio parahaemolytious, rate of accuracy reached to 100%.
Embodiment 4
A kind of kit of Rapid identification vibrio parahaemolytious, including 20 kinds of strong vibrio parahaemolyticus phage mixed liquors and LB
Agar plate.The potency of the strong vibrio parahaemolyticus phage mixed liquor is 109pfu/mL。
The application method of the kit：
Using drop method, the concentration that 25 plants are incubated overnight is 107Cfu/mL various vibrios, 200 μ are drawn with liquid-transfering gun
The LB agar plates center that l is dropped in kit, then LB agar plates are spread evenly across with spreading rod, 20min is stood, is added dropwise 20
Vibrio parahaemolyticus phage 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 parahaemolytious.Identify in 15 plants of vibrios, 9 kinds plaque occur, are accredited as secondary haemolysis arc
Bacterium, rate of accuracy reached to 100%.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, 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 parahaemolyticus phage Rapid identification vibrio parahaemolytious, comprise 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
Flat board；
2) strong vibrio parahaemolyticus phage mixed liquor is added dropwise on the flat board to be checked, obtains identification system；It is described strong
Vibrio parahaemolyticus phage mixed liquor includes more than 10 kinds strong vibrio parahaemolyticus phages；
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 parahaemolytious.
2. according to the method for claim 1, it is characterised in that strong vibrio parahaemolyticus phage mixing described in step 2)
Liquid includes 15~30 kinds of strong vibrio parahaemolyticus phages.
3. according to the method for claim 1, it is characterised in that the coating volume of the step 1) strain to be checked be 100~
200 μ l, it is 20~50 μ l that volume, which is added dropwise, in the strong vibrio parahaemolyticus phage mixed liquor.
4. method according to claim 1 or 2, it is characterised in that the strong vibrio parahaemolytious phagocytosis described in step 2)
The potency of body mixed liquor is 108~1010pfu/mL。
5. according to the method for claim 1, it is characterised in that the acquisition methods of the vibrio parahaemolyticus phage include with
Lower step：
A) vibrio parahaemolyticus phage being separated to and Host Strains vibrio parahaemolytious are mixed, obtain nutrient solution；
B) by the nutrient solution separation of solid and liquid, collection liquid phase component is vibrio parahaemolyticus phage.
6. according to the method for claim 5, it is characterised in that the method for the separation of solid and liquid is centrifugation.
7. according to the method for claim 6, it is characterised 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 parahaemolytious, it is characterised in that mixed including strong vibrio parahaemolyticus phage
Liquid, the strong vibrio parahaemolyticus phage mixed liquor include more than 10 kinds strong vibrio parahaemolyticus phages.
10. kit according to claim 9, it is characterised in that the kit also includes LB agar plates.
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| CN111855753A (en) * | 2020-08-05 | 2020-10-30 | 中国水产科学研究院黄海水产研究所 | On-site detection kit and detection method for vibrio parahaemolyticus in water body |
| CN112080475A (en) * | 2020-07-30 | 2020-12-15 | 扬州大学 | Vibrio parahaemolyticus bacteriophage and application thereof in detection of content of live cells of Vibrio parahaemolyticus pandemic strain |
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| CN112080475A (en) * | 2020-07-30 | 2020-12-15 | 扬州大学 | Vibrio parahaemolyticus bacteriophage and application thereof in detection of content of live cells of Vibrio parahaemolyticus pandemic strain |
| CN111855753A (en) * | 2020-08-05 | 2020-10-30 | 中国水产科学研究院黄海水产研究所 | On-site detection kit and detection method for vibrio parahaemolyticus in water body |
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