CN109576172A - A kind of pre-treating method for GC-MS detection bacterium metabolism group - Google Patents
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Abstract
The invention discloses a kind of pre-treating methods for GC-MS detection bacterium metabolism group, belong to chemical analysis and testing area.The method of the present invention includes the extraction of the culture of bacterium, bacterial solution being quenched with metabolite intracellular；Wherein, bacteria liquid sample is quenched using methanol and sterile aqueous composition, metabolite intracellular is extracted using acetonitrile, isopropanol and sterile aqueous composition, broken extraction mutually is carried out to cell repeatedly with historrhexis's machine combination low temperature ultrasonic, pre-treatment sample is obtained, the substance detected is maximized up to all standing, quantity；The method of the present invention can be used in studying or solving the key scientific problems such as the malicious mechanism of bacterium production, drug resistance generation and control, have a extensive future.
Description
Technical field
Present invention relates particularly to a kind of pre-treating methods for GC-MS detection bacterium metabolism group, belong to chemical analysis
Detection field.
Background technique
Metabolism group is using all metabolic components in the particular organisms sample under qualitative and quantitative qualifications as target, to be
The viewpoint of system biology concentrates all information for illustrating organism metabolism reflection and protein science, transcription group to be complementary to one another,
Become the important means of research biological phenomena together with genomics.
Currently, microbial metabolism group is in developing stage.Since microorganism itself has, system is simple, genome number
Comprehensive feature is understood according to abundant and metabolism network and physiological property, and it has a decisive role in biosystem,
Microbial metabolism group has received widespread attention in microbe research field.Microbial metabolism group has been successfully applied to identify
And the various aspects such as mutation breeding, microbial metabolism engineering, microbial degradation pollutant and Fermentation Engineering.
Bacterial metabolism group is that research bacterium produces in the key scientific problems researchs such as malicious mechanism, drug resistance generation and control not
Can or a scarce ring, therefore the metabolism group in relation to bacterium is also at the startup, for example, Peter Belenky et al. have studied it is anti-
How raw element influences the variation of bacterial metabolism group, lays the foundation for the understanding of its mechanism of action, and enhancing and optimization antibiotic treatment
Method.But current bacterial metabolism group detection method also fails to fully meet the demand of such research, is crushed in pre-treating method
With the insufficient quantity and coverage rate for having seriously affected bacterial metabolism group collection of products of extraction, many has important symbol effect
Metabolic compounds fail and be detected.
Therefore, the efficient pre-treating method for inventing a kind of bacterial metabolism group, for furtheing investigate its physiological metabolism and correlation
Drug resistance, toxicity response mechanism are of great significance.
Summary of the invention
In view of the above shortcomings of the prior art, the present invention is directed to Escherichia coli, salmonella, Staphylococcus aureus
Bacterium, the pre-treating method to bacterium for GC-MS detection bacterium metabolism group are probed into.
It is quenched and reduces rapidly intracellular metabolic enzyme activity, stop metabolic response.Commonly mode, which is quenched, cataclysm
Temperature method and plus organic solvent method.Cataclysm temperature refers to that moment changes sample temperature to -40 DEG C or > 80 DEG C, also has and is gone out using liquid nitrogen
Living, principle is also to reduce rapidly sample temperature to -150 DEG C, inhibits metabolic enzyme activity, collects thallus after 0 DEG C of defrosting.This
Invention research discovery, the degree and quenching time, temperature that the process of being quenched will lead to the leakage i.e. loss of endocellular metabolism object, and reveal
Degree and the organic solvent amount being added etc. are related, and with the addition of buffer, quenching time is reduced, and centrifugal speed is accelerated, and leak journey
Degree can mitigate.Therefore this method is mixed rapidly using pre- cold methanol and bacterium solution, and the method for low-temperature centrifugation reduces leak degree immediately.
The extraction of metabolite is required to extract metabolin to greatest extent, and no skewed popularity does not destroy or change metabolism
The physically or chemically characteristic of object.At present, metabolin extracting method reported in the literature mainly has cold methanol, hot ethanol, perchloric acid
Or alkali, hot methanol, methanol-chloroform mixed liquor and acetonitrile etc..Further study showed that adding in water or methanol and aqueous mixtures
Enter acid acetonitrile and make extractant, recovery rate not only can be improved, but also effectively ribonucleoside triphosphote is inhibited to be degraded into nucleosides and base.
Therefore this method uses acetonitrile, isopropanol and sterile water mixing, is added after extractant and is mutually tied with steel ball historrhexis with ultrasound
The physics mode of conjunction improves the extraction effect of metabolite.
The first purpose of the invention is to provide a kind of pre-treating method for GC-MS detection bacterium metabolism group, institutes
The method of stating includes: the extraction of the culture of bacterium, bacterial solution being quenched with metabolite intracellular；It is described be quenched in the liquid that is quenched be
Methanol and sterile water；The extracting solution of the extraction is acetonitrile, isopropanol and sterile water.
In an embodiment of the present invention, the volume ratio of methanol and sterile water that is quenched in liquid is 3:2~3:1.
In an embodiment of the present invention, the quenching method includes liquid will be quenched mixing with bacterium solution to be quenched, bacterium
Liquid sample is 1:1~1:4 with the volume ratio that liquid is quenched.
In an embodiment of the present invention, in the extracting solution acetonitrile, isopropanol and sterile water volume ratio be (1~
3): (1~3): (1~2).
In an embodiment of the present invention, the extracting method includes being added after mixing extracting solution with somatic cells
Steel ball tissue is crushed, then ultrasonic extraction.
In an embodiment of the present invention, with 121 DEG C of high temperature and high pressure steam autoclave, 20min's sterile water sterilizes
It obtains.
In an embodiment of the present invention, the bacterium includes Escherichia coli, salmonella, staphylococcus aureus.
In an embodiment of the present invention, the quenching method specifically includes:
Obtain methanol and sterile water that liquid is quenched according to the volume mixture of 3:2~3:1, be cooled in advance -20 DEG C it is used below, so
It mixes and is quenched with bacterium solution afterwards.
In an embodiment of the present invention, the bacterial cell intracellular metabolite concentration extracting method specifically includes:
The somatic cells centrifuge tube being quenched is put on ice, the extracting solution of -20 DEG C of pre-coolings is added；Steel ball is added in centrifugation
Guan Zhong is placed in historrhexis's machine and carries out clasmatosis, ultrasonic extraction；Steel ball, centrifugation is sucked out with magnet；Supernatant is taken, it is more
Secondary repetition merges supernatant.
In an embodiment of the present invention, the crusher is 20~40s in 1200~1800rpm, 3~6cycle
Under the conditions of carry out clasmatosis.
In an embodiment of the present invention, the diameter of the steel ball 1-3mm.
In an embodiment of the present invention, the method also includes bacterium solutions to collect, intracellular metabolite concentration is dry and saves.
In an embodiment of the present invention, the method for the Bacteria Culture includes: by single bacterial clump streak inoculation
On nutrient agar, cultivated 12~16 hours (preferably 12h) under the conditions of 37 DEG C；With on aseptic inoculation ring picking plate
Single colony inoculation into nutrient broth fluid nutrient medium, by test tube be placed in 30~37 DEG C of (preferably 37 DEG C) constant-temperature tables 80~
120r/min (preferably 120r/min) culture 12~16 hours (preferably 12h) is turbid to obtain 0.4~0.6 (preferably 0.5) Maxwell
The bacterial solution sample of degree.
In an embodiment of the present invention, the collection method of the bacterial solution includes:
1) sample and it the mixed liquor of liquid is quenched need to quickly be vortexed 5~10s, homogeneous, 4 DEG C of centrifuge 1000~1200g, 3~
After 6min centrifugation, supernatant is abandoned；
2) in same centrifuge tube, repeat it is above-mentioned be quenched with collection step 1~2 time, somatic cells after being quenched.
In an embodiment of the present invention, the store method after the bacterial solution is quenched includes: by the thallus of collection
Sample is centrifuged effective sealed membrane sealing, is placed in ultra low temperature freezer and saves (preferably -80 DEG C).
In an embodiment of the present invention, the dry method of the intracellular metabolite concentration includes: equivalent packing supernatant,
For portion for analyzing, 4~6h is concentrated in portion backup, vacuum refrigeration (preferably 4 DEG C), thoroughly dry, without any in final sample
Water.
In an embodiment of the present invention, the method that the intracellular metabolite concentration saves are as follows: thoroughly after drying, take out vertical
Lid lid is carved, after being centrifuged effective sealed membrane sealing, writes label, low temperature refrigerator is placed in and saves (preferably -20 DEG C) or immediately derivatization.
Second object of the present invention is that the above method is applied to bacterium to produce in terms of malicious mechanism or drug resistance.
The invention has the advantages that:
1, used bacterial solution sample culturing method, it is ensured that bacterium is in logarithmic growth phase in sample bacterium solution,
Vigor is best, and metabolism state is preferable, guarantees that mentioned metabolite result is effective.
2, state and intracellular organic matter situation when quenching method utmostly retains bacteria log growth period
3, collection process has deducted the influence of culture substrate to the greatest extent, and obtains more thallus
4, extracting method ensures that bacterial cell is broken completely, non-targeted to extract metabolite all standing intracellular, quantity most
Bigization avoids omitting important targeting substance.
5, drying may insure that mentioned metabolin property, state in storing process do not change with store method, and
Derivatization effect is not influenced.
Detailed description of the invention
The Metabolism of E. coli profile chromatogram of Fig. 1: 4 DEG C of nutrient agars storage；
The salmonella metabolic profile chromatogram of Fig. 2: 27 DEG C of nutrient agar cultures；
The staphylococcus aureus metabolic profile chromatogram of Fig. 3: 37 DEG C of nutrient agar cultures.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1
Metabolism of E. coli group analysis pre-treating method:
(1) Escherichia coli Liquid Culture:
Escherichia coli are seeded on nutrient agar, cultivate under the conditions of 37 DEG C 12~14 hours, 4 DEG C of storages, with nothing
Single colony inoculation on bacterium oese picking plate is placed in 30~37 DEG C of constant temperature into nutrient broth fluid nutrient medium, by test tube
Shaking table (80~120r/min) cultivates 12~16 hours to obtain the Escherichia coli bacteria liquid sample of 0.4~0.6 Maxwell turbidity；
(2) ice methanol-sterile water is quenched liquid and Bacillus coli cells is quenched:
1. by the liquid that is quenched of bacterium solution cuvette sample and ice, (methanol and sterile water are mixed with 3:2 volume ratio, -20 DEG C of refrigerators
Save pre-cooling) it is placed on mixture of ice and water；
2. liquid is quenched in methanol-water to be added in Escherichia coli bacteria liquid sample, centrifuge tube storage, sample is accumulated with liquid is quenched
For 1:1；
3. sample and the mixed liquor that liquid is quenched quickly are vortexed 5~10s, homogeneous, 4 DEG C of centrifuge 1000~1200g, 3~
After 6min centrifugation, supernatant is abandoned；
4. in same centrifuge tube, repeat 2., 3. step, the somatic cells after being quenched,；- 80 DEG C freeze it is spare.
(3) Escherichia coli substance intracellular extracts:
1. the somatic cells centrifuge tube being quenched is put on ice, the extracting solution (acetonitrile: different of 0.5mL-20 DEG C of pre-cooling is added
Propyl alcohol: water 3:3:2)；
2. the small steel ball of 2~3 pieces of diameter 1mm is added in centrifuge tube, place it in 1200 in historrhexis's machine~
1800rpm, 20~40s, 3~6cycle carries out clasmatosis, 4 DEG C of 10~20min of ultrasonic extraction, and repeats the step 1~2
It is secondary；
3. steel ball is sucked out with magnet, centrifuge tube is put into 4 DEG C of 12000~14000g of centrifuge, is centrifuged 3~6min；
4. supernatant is transferred in new 1.5mL centrifuge tube, repeat it is above-mentioned 1.~4. extraction step and merge supernatant, whirlpool
Rotation mixes；
5. equivalent dispenses supernatant, portion is for analyzing, portion backup；
6. thoroughly after drying, taking out lid lid at once, -20 DEG C of storages for 4 DEG C of 4~6h of freeze concentration of sample vacuum of analysis
It deposits or derivatization immediately.
Processing is obtained into sample and carries out GC-MS detection:
It is detected using Leco Corporation, the U.S. (LECO) Pegasus BT gas-chromatography flight time mass spectrum combined instrument.Chromatostrip
Part: chromatographic column is DB-5MS (Agilent Technologies) fused quartz capillary chromatography column, and specification is 30m (length)
×0.25m (id)×0.25μm(film).Using split sampling mode, 280 DEG C of injector temperature, sampling volume is 1 μ l, is carried
Gas is high-purity helium (purity 99.995%), and flow control uses constant current mode, flow velocity 1ml/min.Temperature program: starting temperature
Degree is 80 DEG C, retains 4min, is then warming up to 300 DEG C with 5 DEG C/min, then retain 10min, 300 DEG C of transmission line temperature；Mass spectrum item
Part: 230 DEG C of ion source temperature, electron bombardment energy 70eV, scanning quality range (50-600) amu, sweep speed 10 opens spectrum
Figure/s, solvent delay time 4min.Chromatography, the mass spectral results that sample detection obtains are as shown in Figure 1, metabolite quantity is 213
It is a.
Embodiment 2
Salmonella metabolic components analyse pre-treating method:
(1) salmonella Liquid Culture
Salmonella, which is seeded on nutrient agar under the conditions of 37 DEG C, to be cultivated 12~18 hours, 4 DEG C of storages, and use is sterile
Test tube is placed in 30~37 DEG C of constant temperature and shaken by the single colony inoculation on oese picking plate into nutrient broth fluid nutrient medium
Bed (80~120r/min) cultivates 12~18 hours to obtain the salmonella bacteria liquid sample of 0.4~0.6 Maxwell turbidity.
(2) ice methanol-sterile water is quenched liquid and salmonella cell is quenched:
1. by the liquid that is quenched of bacterium solution cuvette sample and ice, (methanol and sterile water are mixed with 2:1 volume ratio, -20 DEG C of refrigerators
Save pre-cooling) it is placed on mixture of ice and water；
2. liquid is quenched in methanol-water to be added in Salmonella bacteria liquid sample, centrifuge tube storage, sample and liquid product is quenched it is
1:2；
3. sample and the mixed liquor that liquid is quenched quickly are vortexed 5~10s, homogeneous, 4 DEG C of centrifuge 1000~1200g, 3~
After 6min centrifugation, supernatant is abandoned；
4. in same centrifuge tube, repeat 2., 3. step, be quenched；- 80 DEG C freeze.
(3) salmonella intracellular organic matter extracts
1. the somatic cells centrifuge tube being quenched is put on ice, the extracting solution (acetonitrile: different of 0.5mL-20 DEG C of pre-cooling is added
Propyl alcohol: water 3:2:1)；
2. the small steel ball of 2~3 pieces of diameter 1mm is added in centrifuge tube, place it in 1200 in historrhexis's machine~
1800rpm, 20~40s, 3~6cycle carries out clasmatosis, 4 DEG C of 10~20min of ultrasonic extraction, and repeats the step 1~2
It is secondary；
3. steel ball is sucked out with magnet, centrifuge tube is put into 4 DEG C of 12000~14000g of centrifuge, is centrifuged 3~6min；
4. supernatant is transferred in new 1.5mL centrifuge tube, repeat it is above-mentioned 1.~4. extraction step and merge supernatant, whirlpool
Rotation mixes,；
5. equivalent dispenses supernatant, portion is for analyzing, portion backup；
6. thoroughly after drying, taking out lid lid at once, -20 DEG C of storages for 4 DEG C of 4~6h of freeze concentration of sample vacuum of analysis
It deposits or derivatization immediately.
GC-MS test is carried out referring to method in embodiment 1, gained chromatography, mass spectrum are as shown in Fig. 2, metabolin quantity is 227
It is a.
Embodiment 3
Staphylococcus aureus metabolic components analyse pre-treating method
(1) staphylococcus aureus Liquid Culture
S. aureus Inoculate is cultivated 12~16 hours, use is sterile on nutrient agar under the conditions of 37 DEG C
Test tube is placed in 30~37 DEG C of constant temperature and shaken by the single colony inoculation on oese picking plate into nutrient broth fluid nutrient medium
Bed (80~120r/min) cultivates 12~16 hours to obtain the staphylococcus aureus bacterium solution sample of 0.4~0.6 Maxwell turbidity
Product.
(2) ice methanol-sterile water is quenched liquid and aureus cell is quenched:
1. by the liquid that is quenched of bacterium solution cuvette sample and ice, (methanol and sterile water are mixed with 3:1 volume ratio, -20 DEG C of refrigerators
Save pre-cooling) it is placed on mixture of ice and water；
2. liquid is quenched in methanol-water to be added in staphylococcus aureus liquid sample, sample and liquid is quenched in centrifuge tube storage
Volume is 1:3；
3. sample and the mixed liquor that liquid is quenched quickly are vortexed 5~10s, homogeneous, 4 DEG C of centrifuge 1000~1200g, 3~
After 6min centrifugation, supernatant is abandoned；
4. in same centrifuge tube, repeat 2., 3. step, the somatic cells after being quenched,.- 80 DEG C freeze.
(3) staphylococcus aureus intracellular organic matter extracts:
1. the somatic cells centrifuge tube being quenched is put on ice, the extracting solution (acetonitrile: different of 0.5mL-20 DEG C of pre-cooling is added
Propyl alcohol: water 3:3:1)；
2. the small steel ball of 2~3 pieces of diameter 1mm is added in centrifuge tube, place it in 1200 in historrhexis's machine~
1800rpm, 20~40s, 3~6cycle carries out clasmatosis, 4 DEG C of 10~20min of ultrasonic extraction, and repeats the step 1~2
It is secondary；
3. steel ball is sucked out with magnet, centrifuge tube is put into 4 DEG C of 12000~14000g of centrifuge, is centrifuged 3~6min；
4. supernatant is transferred in new 1.5mL centrifuge tube, repeat it is above-mentioned 1.~4. extraction step and merge supernatant, whirlpool
Rotation mixes；
5. equivalent dispenses supernatant, portion is for analyzing, portion backup；
6. thoroughly after drying, taking out lid lid at once, -20 DEG C of storages for 4 DEG C of 4~6h of freeze concentration of sample vacuum of analysis
It deposits or derivatization immediately.
GC-MS test is carried out referring to method in embodiment 1, gained chromatography, mass spectrum are as shown in figure 3, metabolin quantity is 186
It is a.
Embodiment 4
Salmonella metabolic components analyse pre-treating method:
Referring to embodiment 2, extracting solution is replaced with into the extracting solution 2,3 or 4 in table 2, other conditions are constant, obtained in
Rear somatic cells sample is quenched and carries out GC-MS detection referring to method in embodiment 2, metabolin quantity is as shown in table 2.
Metabolin situation in the sample of the different extracting solution preparations of table 2
| Extracting solution ingredient | Extracting solution component ratio | Metabolin quantity | |
| Extracting solution 2 | - 20 DEG C of pre- cold methanols and sterile water | 2:1 | 207 |
| Extracting solution 3 | Methanol and chloroform | 2:1 | 201 |
| Extracting solution 4 | Cold perchloric acid | 0.25M/mL | 202 |
Embodiment 5
Salmonella metabolic components analyse pre-treating method:
Referring to embodiment 2, breaking method when extracting is changed to liquid nitrogen grinding, other conditions are constant, sample is prepared,
GC-MS detection is carried out, as a result metabolin quantity is 189.
Reference examples 1
Different quenching methods are investigated:
Referring to embodiment 2, liquid will be quenched replacing in table 1 liquid 2 or 3 is quenched, other conditions are constant, wherein after processing
To sample in detect by GC-MS, metabolin situation is as shown in table 1.
Metabolin situation in the sample of liquid preparation is quenched in 1 difference of table
| Liquid ingredient is quenched | Liquid component volume ratio is quenched | Metabolin quantity (a) | |
| Embodiment 2 | Methanol and sterile water | 2:1 | 227 |
| Liquid 2 is quenched | Methanol and chloroform | 2:1 | 95 |
| Liquid 3 is quenched | 90 DEG C of hot ethanols | / | 97 |
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of pre-treating method for GC-MS detection bacterium metabolism group, which is characterized in that the described method includes: bacterium
Culture, bacterial solution the extraction being quenched with metabolite intracellular；It is described be quenched be quenched liquid be methanol and sterile water, wherein
The volume ratio of methanol and sterile water is 3:2~3:1.
2. the method according to claim 1, wherein the quenching method includes that liquid will be quenched to be mixed into bacterium solution
Row is quenched, and wherein bacteria liquid sample and the volume ratio that liquid is quenched are 1:1~1:4.
3. the method according to claim 1, wherein the extracting solution of the extraction is acetonitrile, isopropanol and sterile
Water.
4. according to the method described in claim 3, it is characterized in that, the volume ratio of acetonitrile, isopropanol and sterile water is (1~3):
(1~3): (1~2).
5. method according to claim 1 to 4, which is characterized in that the extracting method includes by extracting solution and thallus
After mixing with cells, steel ball is added and is crushed, then ultrasonic extraction.
6. the method according to claim 1, wherein the quenching method specifically includes:
Methanol and sterile water are mixed to get according to volume ratio, liquid are quenched, be cooled in advance -20 DEG C it is used below, it is then mixed with bacterium solution
Conjunction is quenched.
7. the method according to claim 1, wherein the bacterial cell intracellular metabolite concentration extracting method is specific
Include:
The extracting solution that somatic cells are pre-chilled with -20 DEG C or less is mixed, steel ball is added in centrifuge tube, is placed in historrhexis's machine
Clasmatosis is carried out, then ultrasonic extraction, steel ball, centrifugation is sucked out, takes supernatant, is repeated several times, merges supernatant.
8. the method according to claim 1, wherein the bacterium includes Escherichia coli, salmonella, golden yellow
Staphylococcus.
9. -8 any method according to claim 1, which is characterized in that the method also includes bacterium solutions to collect, generation intracellular
It thanks to product drying and saves.
10. application of any the method for claim 1-9 in terms of bacterium produces malicious mechanism or drug resistance.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111650292A (en) * | 2020-04-20 | 2020-09-11 | 广东省测试分析研究所（中国广州分析测试中心） | Enrichment and detection method for non-targeted volatile metabolite of pathogenic escherichia coli and application thereof |
| CN112540133A (en) * | 2020-11-02 | 2021-03-23 | 暨南大学 | Detection and analysis method for marine microalgae intracellular metabolome |
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