CN106636073A - Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture - Google Patents
Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture Download PDFInfo
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- CN106636073A CN106636073A CN201710046863.6A CN201710046863A CN106636073A CN 106636073 A CN106636073 A CN 106636073A CN 201710046863 A CN201710046863 A CN 201710046863A CN 106636073 A CN106636073 A CN 106636073A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a preparation method of a pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture. The preparation method comprises the following steps: carrying out shaking-bed culture on three test tubes with a liquid culture medium at 28 DEG C for one night; taking out the three test tubes the next day; taking one test tube as a control group and taking two test tubes as experiment groups; opening a clean worktable; firstly, carrying out ultraviolet disinfection for 20min and then putting the test tubes into the clean worktable; selecting one test tube with bacteria, which has a relatively great concentration; sucking 1ml of a bacterium culture solution into a prepared PCR (Polymerase Chain Reaction) tube by utilizing a pipette; adding an equal amount of clean water into the other PCR tube, so as to conveniently symmetrize in a centrifuging process. The preparation method disclosed by the invention is simple and has a simple preparation effect; the template can be used for effectively detecting vibrio infection in a culture water area or tissues in cultured prawns; water quality regulation and control can be carried out in advance, prawn vibrio diseases are prevented early and large-scale outbreak of diseases is prevented and controlled, so that healthy culture of the prawns is effectively improved.
Description
Technical field
The present invention relates to Vibrio harveyi technical field, pathogenic Kazakhstan Vickers in specifically a kind of prawn culturing
The preparation method of vibrios DNA profiling.
Background technology
The Major Diseases of prawn (Crustin, Marsupenaeus japonicus, Environment of Litopenaeus vannamei Low) cultivation at present are Leucoplakia synthesis
Disease (WSSV), in the shrimp samples sampling Detection of 124 batches, WSSV positive rates are more than 80%；Next to that vibrio infection is red
Leg disease, and Vibrio harveyi is wherein topmost virulence factor, in the shrimp samples sampling Detection of 209 batches, breathes out Vickers arc
The positive rate of bacterium is more than 70%.
Vibrio harveyi not only can directly cause the prawn for cultivating to be caused a disease, and prawn appendage occur and redden, and swimmeret becomes earliest
Red, step and uropodium are also in cerise, and the gill area of carapace is in yellow, and apocleisis, power weakens, and individuality is become thin phenomenon, can also be fast
Speed causes the prawn in same breeding water body to infect and break out large-scale red leg disease, causes prawn quick and substantial amounts of death
Phenomenon, endangers very huge.
The content of the invention
Present invention aims to the defect and deficiency of prior art, there is provided pathogenic Kazakhstan in a kind of prawn culturing
The preparation method of Vickers vibrios DNA profiling.
For achieving the above object, the technical solution used in the present invention is：
A kind of preparation method of pathogenic Vibrio harveyi DNA profiling in prawn culturing, its step is as follows：
1st, by three test tubes with fluid nutrient medium, shaking table culture is overnight at 28 DEG C；
2nd, three test tubes are taken out within second day, a control group, two experimental groups open clean bench, purple is carried out first
Outer sterilization 20min is latter to be put into wherein together；
3rd, the larger test tube that carries disease germs of concentration ratio is chosen, 1ml inoculums is drawn with liquid-transfering gun and is ready in advance
PCR pipe in, and again in another PCR pipe plus equivalent clear water in order to be centrifuged when it is symmetrical；
4th, above-mentioned two PCR pipe is placed in supercentrifuge, 5min is centrifuged, rotating speed is 1200r/min, abandons supernatant
(slowly being drawn with the less liquid-transfering gun of range)；
5th, 100ul sterilized waters are added, heating water bath 10min in 100 DEG C of water-baths is placed in；
6th, and then ice bath 2min；
7th, it is placed on again in supercentrifuge, rotating speed is 12000r/min, 5min, takes supernatant standby；
8th, it is placed in one 20 DEG C of refrigerators and saves backup.
Beneficial effects of the present invention are：Preparation method is simple, and preparation effect is good, can effectively detect Cultivated water and support
The vibrio infection in prawn tissue is grown, regulating and controlling water quality is carried out in advance, prevention vibriosis penaeus early prevent and treat scale outburst disease
Disease, so as to effectively improve the healthy aquaculture of prawn.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is right below in conjunction with specific embodiment
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
A kind of preparation method of pathogenic Vibrio harveyi DNA profiling in prawn culturing, its step is as follows：
1st, by three test tubes with fluid nutrient medium, shaking table culture is overnight at 28 DEG C；
2nd, three test tubes are taken out within second day, a control group, two experimental groups open clean bench, purple is carried out first
Outer sterilization 20min is latter to be put into wherein together；
3rd, the larger test tube that carries disease germs of concentration ratio is chosen, 1ml inoculums is drawn with liquid-transfering gun and is ready in advance
PCR pipe in, and again in another PCR pipe plus equivalent clear water in order to be centrifuged when it is symmetrical；
4th, above-mentioned two PCR pipe is placed in supercentrifuge, 5min is centrifuged, rotating speed is 1200r/min, abandons supernatant
(slowly being drawn with the less liquid-transfering gun of range)；
5th, 100ul sterilized waters are added, heating water bath 10min in 100 DEG C of water-baths is placed in；
6th, and then ice bath 2min；
7th, it is placed on again in supercentrifuge, rotating speed is 12000r/min, 5min, takes supernatant standby；
8th, it is placed in one 20 DEG C of refrigerators and saves backup.
The advantage of this specific embodiment：Preparation method is simple, and it is good to prepare effect, can effectively detect Cultivated water and
Vibrio infection in cultured prawn tissue, carries out in advance regulating and controlling water quality, prevention vibriosis penaeus early, prevents and treats scale outburst
Disease, so as to effectively improve the healthy aquaculture of prawn.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity those skilled in the art should
Using specification as an entirety, the technical scheme in each embodiment can also Jing it is appropriately combined, form those skilled in the art
Understandable other embodiment.
Claims (1)
1. in a kind of prawn culturing pathogenic Vibrio harveyi DNA profiling preparation method, it is characterised in that：Step is as follows：
(1), by three test tubes with fluid nutrient medium, shaking table culture is overnight at 28 DEG C；
(2) three test tubes, are taken out within second day, a control group, two experimental groups are opened clean bench, carried out first ultraviolet
Sterilization 20min is latter to be put into wherein together；
(3) the larger test tube that carries disease germs of concentration ratio, is chosen, 1ml inoculums is drawn in preprepared with liquid-transfering gun
It is in PCR pipe and symmetrical when adding equivalent clear water in order to be centrifuged in another PCR pipe again；
(4), above-mentioned two PCR pipe is placed in supercentrifuge, 5min is centrifuged, rotating speed is 1200r/min, abandoned supernatant and (use
The less liquid-transfering gun of range is slowly drawn)；
(5) 100ul sterilized waters, are added, heating water bath 10min in 100 DEG C of water-baths is placed in；
(6), and then ice bath 2min；
(7), it is placed on again in supercentrifuge, rotating speed is 12000r/min, 5min, takes supernatant standby；
(8), it is placed in one 20 DEG C of refrigerators and saves backup.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710046863.6A CN106636073A (en) | 2017-01-20 | 2017-01-20 | Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture |
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| CN201710046863.6A CN106636073A (en) | 2017-01-20 | 2017-01-20 | Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113652376A (en) * | 2021-09-14 | 2021-11-16 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | Pathogenic vibrio harveyi and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838691A (en) * | 2010-02-02 | 2010-09-22 | 中国海洋大学 | Kit for rapidly detecting fish Vibrio harveyi PCR and using method |
| CN102108402A (en) * | 2010-12-14 | 2011-06-29 | 陈吉刚 | Kit and detection method for detecting vibrio harveyi |
| CN105296617A (en) * | 2015-10-10 | 2016-02-03 | 宁波大学 | Gene chip for detecting vibrio harveyi colony and using method of gene chip |
-
2017
- 2017-01-20 CN CN201710046863.6A patent/CN106636073A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101838691A (en) * | 2010-02-02 | 2010-09-22 | 中国海洋大学 | Kit for rapidly detecting fish Vibrio harveyi PCR and using method |
| CN102108402A (en) * | 2010-12-14 | 2011-06-29 | 陈吉刚 | Kit and detection method for detecting vibrio harveyi |
| CN105296617A (en) * | 2015-10-10 | 2016-02-03 | 宁波大学 | Gene chip for detecting vibrio harveyi colony and using method of gene chip |
Non-Patent Citations (3)
| Title |
|---|
| 徐芝亮: "《哈维氏弧菌遗传多样性分析及毒力相关基因的检测》", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 王刚等: "《哈维氏弧菌和白斑综合症病毒对墨吉明对虾仔虾的致病性》", 《热带生物学报》 * |
| 程蝶等: "《环介导等温扩增联合横向流动试纸可视化检测哈维氏弧菌的研究》", 《生物技术通报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113652376A (en) * | 2021-09-14 | 2021-11-16 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | Pathogenic vibrio harveyi and application thereof |
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Application publication date: 20170510 |