CN104560813A - <15>N SIP (stable isotope probing) vibrio parahaemolyticus culture medium and cultivation method thereof - Google Patents
<15>N SIP (stable isotope probing) vibrio parahaemolyticus culture medium and cultivation method thereof Download PDFInfo
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Abstract
The invention discloses a <15>N SIP (stable isotope probing) vibrio parahaemolyticus culture medium and a cultivation method thereof. A formula of the culture medium comprises components as follows: 1-5 g/L of a nitrogen source, 5-18 g/L of a carbon source, 0.5-2.0 g/L of KH2PO4, 0.6-2.0 g/L of K2HPO4, 0.1-1.0 g/L of MgSO4, 10-30 g/L of NaCl and 1L of distilled water, the pH is in a range from 7.0 to 7.5, and nitrogen atoms of the nitrogen source are labeled by adopting <15>N. The cultivation method of the <15>N SIP vibrio parahaemolyticus comprises steps as follows: 1), continuously and repeatedly cultivating the vibrio parahaemolyticus through transferring; 2), verifying the labeling efficiency of protein nitrogen atoms in labeled vibrio parahaemolyticus cells with LC-MS/MS (high-performance liquid chromatography-tandem mass spectrometry). The process is simple and reasonable, the culture medium has simple components and is low in cost, and the novel culture medium preparation method is provided for industrial preparation of the <15>N SIP vibrio parahaemolyticus culture medium.
Description
Technical field
The present invention relates to one
15n stable isotope mark Vibrio parahaemolyticus substratum and method of cultivation thereof.
Background technology
In ocean environment, most vibrios is modal bacteria types, and they maintain the normal marine eco-environment, but some vibrios is harmful to the mankind and marine animal.Nineteen ninety-five " U.S. clinical microorganism handbook " sixth version specify that in vibrios, pathogenic bacterium are 12 kinds, in these 12 kinds of pathogenic bacterium, the most important thing is vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus.The FDA of the U.S., the NMKL in Europe, ministry of Health of China has all formulated corresponding detection method.Kinds of pathogenic vibrio associated diseases comprises gastro-enteritis, wound infection etc., and the food poisoning that such as Vibrio parahaemolyticus causes propagated through cooking sea-food or salt curing food improperly.Common is jellyfish, ocean fish, extra large shrimp and various shellfish, because foodstuff container or chopping block are given birth to ripe in after this bacterium of pollution, can poison by food.
Current Vibrio parahaemolyticus (Vibrio parahaemolyticus) accounts for first of bacterial food poisoning, and in the food poisoning case of coastland in recent years, this bacterium has become primary cause of disease.The virulence factor of Vibrio parahaemolyticus has hemolysin, lipopolysaccharides, extracellular protease, urease and III type excretory system etc.Under various environmental stimulus, as low temperature (cold chain food), high salt (cure food), high temperature, acid, alkali etc., Vibrio parahaemolyticus is in faulted condition, pathogenicly changes.How effectively the impaired kinds of pathogenic vibrio of monitoring, especially Vibrio parahaemolyticus survivor state, assess consumer in various environment are finally exposed to possibility in the kinds of pathogenic vibrio such as Vibrio parahaemolyticus and have important directive significance.
Cold labeling (Stable Isotope Probing, SIP) technology with the function of its spike, instruction and judgement and detect fast, result is accurate etc., and feature becomes one of important research means understanding protein, nucleic acid dynamic change in pathogenic bacterium damaged process.Cold labeling technology mainly comprises two kinds of different routes: one is called metabolic marker method, is to add when cultivating with isotope-labeled amino acid whose form; Another kind is called chemical labeling method, is after proteins extraction, and some functional groups and the reagent generation chemical reaction containing cold labeling of the protein before, during and after enzymolysis, wherein the former reports less, and the latter is widely used.The former generally adopts and must cultivate microorganism by amino acid whose substratum containing heavy stable isotope type, but no matter adopts microbe fermentation method or adopt the amino acid whose method of organic chemical synthesis cold labeling, and cost is high, complex process; After the latter adopts proteins extraction, the reagent generation chemical reaction of protein functional groups (as peptide section) and cold labeling, side reaction is many, complex process.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of
15the method of N stable isotope mark Vibrio parahaemolyticus substratum.Vibrio parahaemolyticus repeatedly to be transferred cultivation by the mark substratum of this invention, and in mycetocyte, almost all nitrogen-atoms is replaced as stable isotope
15n, wherein in mass spectroscopy particularly LC-MS/MS, easy and verify safely.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Of the present invention
15the substratum of N stable isotope mark Vibrio parahaemolyticus, the formula of described substratum is: comprise that nitrogenous source is 1-5g/L, carbon source is 5-18g/L, KH
2pO
4for 0.5-2.0g/L, K
2hPO
4for 0.6-2.0g/L, MgSO
4for 0.1-1.0g/L, NaCl are 10-30g/L, distilled water 1L, pH7.0 ~ 7.5, the nitrogen-atoms of nitrogenous source adopts
15n marks.
Preferably, the formula of described substratum is: comprise that nitrogenous source is 2g/L, carbon source is 10g/L, KH
2pO
4for 0.6g/L, K
2hPO
4for 0.9g/L, MgSO
4for 0.2g/L, NaCl are 20g/L, distilled water 1L, pH7.0 ~ 7.5, the nitrogen-atoms of nitrogenous source adopts
15n marks.
Preferably, nitrogenous source is (NH
4)
2sO4, NH
4one in Cl and urea.
Preferably, carbon source is the one in glucose and sodium acetate.
Present invention also offers a kind of cultivation
15n marks the method for Vibrio parahaemolyticus, and described method comprises:
1) Vibrio parahaemolyticus is cultivated in continuous several times switching:
(1) carrying out first time in Vibrio parahaemolyticus inoculation to 3% sodium-chlor basic peptone water increases bacterium, cultivates 1 ~ 2 day under 25 ~ 36 DEG C of shaking table conditions;
(2) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 5 ~ 10 rings increasing bacterium in step (1)
15carry out second time in N cold labeling substratum and increase bacterium, cultivate 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
(3) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 1 ~ 3 ring increasing bacterium in step (2)
15carrying out third time in N cold labeling substratum increases bacterium, cultivates 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
(4) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 1 ~ 3 ring increasing bacterium in step (3)
15carry out the 4th time in N cold labeling substratum and increase bacterium, cultivate 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
Described 3% sodium-chlor basic peptone water formula is:
Peptone 10g/L, sodium-chlor 30g/L, distilled water 1L, pH8.5 ~ 9.0,121 DEG C of autoclaving 10min.
2) labeling effciency of protein nitrogen atom in the Vibrio parahaemolyticus mycetocyte of LC-MS/MS verification mark:
(1) reach after required bacterium amount until above-mentioned cultivation, with 10,000 rev/min is centrifugal 5 ~ 10 minutes, collect step (4) in increase the Vibrio parahaemolyticus mycetocyte of bacterium the 4th time;
(2) adopt ultrasonication method to extract total protein the Vibrio parahaemolyticus mycetocyte collected, use bradford method to carry out quantitatively to total protein;
By total protein quantitatively after get 5 μ g, add trypsin digestion after reductive alkylation;
(4) by enzymolysis solution 10000r/min, 4 DEG C centrifugal after get supernatant liquor, ZipTip desalination, after centrifuge concentrator drying, 0.1% formic acid solution redissolves, and gets supernatant liquor and inject LC-MS/MS instrumental analysis after high speed centrifugation.
Vibrio parahaemolyticus repeatedly to be transferred cultivation by the mark substratum of this invention, and in mycetocyte, almost all nitrogen-atoms is replaced as stable isotope
15n, wherein in mass spectroscopy particularly LC-MS/MS, easy and verify safely.The method technique advantages of simple, nutrient media components is simple, and cost is low, is preparation of industrialization
15n marks Vibrio parahaemolyticus substratum and provides new medium preparation method.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1-A is the total ion chromatogram of Vibrio parahaemolyticus ATCC17802 total protein; Fig. 1-B is the one-level color atlas of a peptide section of lipoic dehydrogenase (Lipoic acid amine dehydrogenase) in Vibrio parahaemolyticus ATCC17802.
Embodiment
Embodiment 1
Embodiment 1:
Vibrio parahaemolyticus bacterial strain is Vibrio parahaemolyticus (Vibrio parahaemolyticus) ATCC 17802.
1)
15the preparation of N cold labeling substratum:
In the present embodiment
15n cold labeling substratum is by NH
4cl, glucose, KH
2pO
4, K
2hPO4, MgSO
4, NaCl, distilled water composition; Wherein NH
4cl content is 2g/L, and glucose content is 10g/L, KH
2pO
4content is 0.6g/L, K
2hPO4 content is 0.9g/L, MgSO
4content is 0.2g/L, NaCl content is 20g/L, and distilled water content is 1L; The pH value of substratum is 7.0 ~ 7.5;
15nH in N cold labeling substratum
4the nitrogen-atoms of Cl adopts
15n carries out marking (abundance, 98%).
2) Vibrio parahaemolyticus is cultivated in continuous several times switching:
(1) Vibrio parahaemolyticus ATCC17802 is inoculated in 3% sodium-chlor basic peptone water and carries out first time and increase bacterium, under 30 DEG C of shaking table conditions, cultivate 18h.Wherein 3% sodium-chlor basic peptone water formula is: peptone 10g/L, sodium-chlor 30g/L, distilled water 1L, pH8.5 ~ 9.0,121 DEG C of autoclaving 10min.
(2) adopt transfering loop 10 rings of being transferred by the Vibrio parahaemolyticus ATCC17802 increasing bacterium in step (1) to arrive
15carry out second time in N cold labeling substratum and increase bacterium, under 36 DEG C of shaking table conditions, cultivate 40h.
(3) adopt transfering loop 3 rings of being transferred by the Vibrio parahaemolyticus ATCC17802 increasing bacterium in step (2) to arrive
15carrying out third time in N cold labeling substratum increases bacterium, under 36 DEG C of shaking table conditions, cultivate 36h.
(4) adopt transfering loop 2 rings of being transferred by the Vibrio parahaemolyticus ATCC17802 increasing bacterium in step (3) to arrive
15carry out the 4th time in N cold labeling substratum and increase bacterium, under 36 DEG C of shaking table conditions, cultivate 24h.
3) labeling effciency of protein nitrogen atom in the Vibrio parahaemolyticus mycetocyte of LC-MS/MS verification mark:
(1), with 10,000 rev/min is centrifugal 5 ~ 10 minutes, collect step (4) in increase the Vibrio parahaemolyticus ATCC17802 mycetocyte of bacterium the 4th time.
(2) adopt ultrasonication method to extract total protein the Vibrio parahaemolyticus ATCC17802 mycetocyte collected, use bradford method to carry out quantitatively total protein.
By total protein quantitatively after get 5 μ g, add trypsin digestion after reductive alkylation.
(4) by enzymolysis solution 10000r/min, 4 DEG C centrifugal after get supernatant liquor, ZipTip desalination, after centrifuge concentrator drying, 0.1% formic acid solution redissolves, and gets supernatant liquor and inject LC-MS/MS instrumental analysis after high speed centrifugation.
Liquid phase: Thermo Scientific Easy-nLC 1000;
Chromatographic condition is: chromatographic column: Easy-spray column (C18,2 μm,
75 μm of x 50cm); Applied sample amount: equal 10 μ L, mobile phase A: 0.1%Formic acid in water; B:0.1%Formic acid in Acetonitrile.
Chromatogram gradient:
| Time (min) | Time length (min) | ％B |
| 0 | 0 | 3 |
| 15:0 | 15 | 8 |
| 16:0 | 1 | 8 |
| 51:0 | 35 | 18 |
| 66:0 | 15 | 28 |
| 67:0 | 1 | 95 |
| 75:0 | 8 | 95 |
Flow velocity: 400nL/min.
Mass spectrometric detection condition: track hydrazine mass spectrograph Thermo fisher Orbitrap Velos
Positive ion scan mode, first mass spectrometric sweep limit m/z 375 ~ 1300,250ms, MS1resolution is 60000, selects 50 the highest peaks of first order spectrum to carry out secondary scanning, sweep limit m/z 375 ~ 1300, collision voltage: 35CID.
(5) by changing the ratio optimization chromatographic separation condition of mobile phase A and Mobile phase B.Under the LC-MS/MS condition optimized, finally obtain the total ion chromatogram (Fig. 1-A) of Vibrio parahaemolyticus ATCC17802 total protein, get a quilt at random
15the albumen (Lipoic acid amine dehydrogenase) of N cold labeling, the one-level color atlas (Fig. 1-B) of a peptide section of this albumen calculates checking through molecular weight and can reach a conclusion: the nitrogen-atoms in this albumen by
14n is replaced as stable isotope
15n.
Claims (6)
1. one kind
15the substratum of N stable isotope mark Vibrio parahaemolyticus, it is characterized in that, the formula of described substratum is: comprise that nitrogenous source is 1-5 g/L, carbon source is 5-18 g/L, KH
2pO
4for 0.5-2.0 g/L, K
2hPO
4for 0.6-2.0 g/L, MgSO
4for 0.1-1.0 g/L, NaCl are 10-30 g/L, distilled water 1L, pH7.0 ~ 7.5, the nitrogen-atoms of nitrogenous source adopts
15n marks.
2. substratum according to claim 1, is characterized in that, the formula of described substratum is: comprise that nitrogenous source is 2 g/L, carbon source is 10 g/L, KH
2pO
4be 0.6 g/L, K
2hPO
4be 0.9 g/L, MgSO
4be 0.2 g/L, NaCl be 20 g/L, distilled water 1L, pH7.0 ~ 7.5, the nitrogen-atoms of nitrogenous source adopts
15n marks.
3. substratum according to claim 1, is characterized in that, nitrogenous source is (NH
4)
2sO4, NH
4one in Cl and urea.
4. substratum according to claim 1, is characterized in that, carbon source is the one in glucose and sodium acetate.
5. a cultivation
15n marks the method for Vibrio parahaemolyticus, and it is characterized in that, described method comprises:
1) Vibrio parahaemolyticus is cultivated in continuous several times switching:
(1) carrying out first time in Vibrio parahaemolyticus inoculation to 3% sodium-chlor basic peptone water increases bacterium, cultivates 1 ~ 2 day under 25 ~ 36 DEG C of shaking table conditions;
(2) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 5 ~ 10 rings increasing bacterium in step (1)
15carry out second time in N cold labeling substratum and increase bacterium, cultivate 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
(3) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 1 ~ 3 ring increasing bacterium in step (2)
15carrying out third time in N cold labeling substratum increases bacterium, cultivates 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
(4) adopt transfering loop to be arrived by Vibrio parahaemolyticus switching 1 ~ 3 ring increasing bacterium in step (3)
15carry out the 4th time in N cold labeling substratum and increase bacterium, cultivate 1 ~ 2 day under 30 ~ 36 DEG C of shaking table conditions;
2) labeling effciency of protein nitrogen atom in the Vibrio parahaemolyticus mycetocyte of LC-MS/MS verification mark:
(1) reach after required bacterium amount until above-mentioned cultivation, with 10,000 rev/min is centrifugal 5 ~ 10 minutes, collect step (4) in increase the Vibrio parahaemolyticus mycetocyte of bacterium the 4th time;
(2) adopt ultrasonication method to extract total protein the Vibrio parahaemolyticus mycetocyte collected, use bradford method to carry out quantitatively to total protein;
By total protein quantitatively after get 5 μ g, add trypsin digestion after reductive alkylation;
(4) by enzymolysis solution 10000 r/ min, 4 DEG C centrifugal after get supernatant liquor, ZipTip desalination, after centrifuge concentrator drying, 0.1% formic acid solution redissolves, and gets supernatant liquor and inject LC-MS/MS instrumental analysis after high speed centrifugation.
6. method according to claim 4, is characterized in that, described 3% sodium-chlor basic peptone water formula is: peptone 10 g/L, sodium-chlor 30 g/L, distilled water 1L, pH8.5 ~ 9.0,121 DEG C of autoclaving 10 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107389812A (en) * | 2017-07-07 | 2017-11-24 | 山东出入境检验检疫局检验检疫技术中心 | With the method for vibrio parahaemolytious in internal standard method protein group quantitative determination aquatic products |
| CN107793209A (en) * | 2016-08-30 | 2018-03-13 | 泰生科技香港有限公司 | Special culture media, the preparation method and the usage of stable isotope labeled plant |
Citations (1)
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| CN102703566A (en) * | 2012-05-31 | 2012-10-03 | 上海市疾病预防控制中心 | Plate kit for identifying vibrio parahemolyticus toxigenic strain, and preparation and using methods for plate kit |
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2014
- 2014-12-30 CN CN201410841379.9A patent/CN104560813A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102703566A (en) * | 2012-05-31 | 2012-10-03 | 上海市疾病预防控制中心 | Plate kit for identifying vibrio parahemolyticus toxigenic strain, and preparation and using methods for plate kit |
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|---|
| BLACKSTONE G M等: "Detection of pathogenic Vibrio parahaemolyticus in oyster enrichments by real time PCR", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| 单洪伟等: "不同C:N条件下一株溶藻弧菌对不同种类氮源的吸收特性", 《中国水产科学》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107793209A (en) * | 2016-08-30 | 2018-03-13 | 泰生科技香港有限公司 | Special culture media, the preparation method and the usage of stable isotope labeled plant |
| CN107389812A (en) * | 2017-07-07 | 2017-11-24 | 山东出入境检验检疫局检验检疫技术中心 | With the method for vibrio parahaemolytious in internal standard method protein group quantitative determination aquatic products |
| CN107389812B (en) * | 2017-07-07 | 2020-02-25 | 青岛海关技术中心 | Method for quantitatively determining vibrio parahaemolyticus in aquatic product by using proteome of internal standard method |
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