CN110029082A - A kind of preparation and its detection method of the Escherichia coli mediated based on 1,4-benzoquinone - Google Patents
A kind of preparation and its detection method of the Escherichia coli mediated based on 1,4-benzoquinone Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 28
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Abstract
The invention discloses the preparations and its detection method of a kind of Escherichia coli mediated based on 1,4-benzoquinone, it is related to biologic medical detection field, wild-type e. coli (WT) is inoculated into a series of conical flasks with dual antibiotic concentration gradient and cultivates by preparation method, and carries out oscillation bath；Then the OD of each conical flask is measured600Value, the bacterial strain breeding with highest antibiotic concentration for again growing wild-type e. coli (WT) is into the next round antibiotic resistance culture with higher antibiotic concentration gradient, after above step carries out 5 wheels, low antibiotic resistance Escherichia coli are collected；After 10 wheel selections, high antibiotic resistant E. coli is collected.Using the colorimetric method and electrochemical assay mediated based on 1,4-benzoquinone can specificity and selectivity detection and calibration Escherichia coli, while can also potentially distinguish antibiotic resistance Escherichia coli.Simple and sensitive of the present invention, convenient effective, low in cost, suitable for large-scale promotion and production has biggish application prospect.
Description
Technical field
The present invention relates to biologic medical detection field more particularly to a kind of preparations of the Escherichia coli mediated based on 1,4-benzoquinone
And its detection method.
Background technique
The presence of Escherichia coli is considered as the index of water pollution, and the Escherichia coli of high concentration can lead to serious disease,
Such as diarrhea, urinary tract infections, meningitis, anaemia and kidney failure.Conventional bacteria detection method (including bacterial cell separation, culture and
Count etc.) it obtains result and typically at least needs two weeks, and it is longer when determining resistance of the sample to certain antibiotics；Laboratory
In common method (including polymerase chain reaction, surface plasma body resonant vibration, enzyme linked immunosorbent assay (ELISA), surface-enhanced Raman
Scattering, mass spectrum, microarray and biosensor etc.) though it is accurate, sensitive, it more expensive, time-consuming, large labor intensity and needs
Special instrument leads to the bad adaptability of field assay.
Detection based on colorimetric is capable of providing simple and visual analysis without complicated operation or instrument；Existing base at present
In several different principles of electrochemical techniques, to the simple of bacterium, quick and Sensitive Detection wide coverage.These method energy
Enough sensitive and selectively detection bacterium, but need expensive modification, including temperature sensitivity enzyme, nano material synthesis and superfluous
Long analysis program, these all limit practical utilization.1,4-benzoquinone (BQ) is well-known lipophilicity redox mediators, can
For electrochemica biological sensor, for measuring toxicity, glucose and substrate oxidation activity.It is used in bio-sensor system
The recognition component that 1,4-benzoquinone mediates has become mature and universal.Large intestine is carried out using 1,4-benzoquinone however, there is presently no documents
The detection of bacillus quantity and antibiotic resistance analysis.
Therefore, those skilled in the art are dedicated to developing a kind of innovation, cheap, E. coli detection side rapidly and simply
Method can be used to quantify Escherichia coli and distinguish its antibiotic resistant.
Summary of the invention
In view of the above drawbacks of the prior art, simple the technical problem to be solved by the present invention is to conventional identification method but
Time-consuming, insensitive, and experimental identification method is sensitive but costly, time-consuming, large labor intensity.
To achieve the above object, the present invention provides a kind of antibiotic resistance Escherichia coli preparation method, the preparation methods
The following steps are included:
Wild-type e. coli (WT) is inoculated into a series of tapers with dual antibiotic concentration gradient by step 1-1
It is cultivated in flask, and carries out oscillation bath；
The OD of each conical flask after step 1-2, measuring process 1-1600It is worth (light absorption value of the solution at 600nm wavelength), with
Measure the concentration of wild-type e. coli (WT) culture solution；
Step 1-3, the bacterial strain with highest antibiotic concentration for growing wild-type e. coli (WT) are bred to having
In the next round antibiotic resistance culture of higher antibiotic concentration gradient；
Step 1-4 collects low antibiotic resistance Escherichia coli after step 1-3 carries out 5 wheel selections；
Step 1-5 collects high antibiotic resistant E. coli after step 1-3 carries out 10 wheel selections.
Further, the initial concentration of dual antibiotic concentration gradient described in step 1-1 is minimum inhibitory concentration
50%.
Further, the temperature that bath is vibrated in step 1-1 is 37 DEG C, and oscillation revolving speed is 150rpm.
The present invention also provides a kind of detection method of antibiotic resistance Escherichia coli mediated based on 1,4-benzoquinone, this method
For colorimetric method, specifically includes the following steps:
Tested bacteria is placed in LB culture medium by step 2-1, stays overnight the bacterial culture growth；
Step 2-2, the bacterial cultures that step 2-1 is obtained are centrifuged；
Step 2-3 is washed bacterial cultures in step 2-2 three times with phosphate buffered saline solution, and by remaining bacterium
Sediment is suspended in phosphate buffered saline solution for use；
It is dense to add difference obtained in 100 μ L 1,4-benzoquinone and 100 μ L step 2-3 in 96 hole microtest plates by step 2-4
The bacterial suspension of degree；
Step 2-5 records the color change of 96 orifice plates after reaction 1 hour.
Further, the growth time of bacterial cultures is 12 hours in step 2-1, and temperature is 37 DEG C, and concussion revolving speed is
150rpm。
Further, centrifugation rate is 3600rpm in step 2-2, and centrifugation time is 10 minutes.
Further, the concentration of 1,4-benzoquinone is 6mM in step 2-4, the concentration of each bacterial suspension is respectively 1 ×
109CFU/mL,1×108CFU/mL,1×107CFU/mL,1×106CFU/mL,1×105CFU/mL,1×104CFU/mL,1×
103CFU/mL。
The present invention also provides a kind of Resistance detection method of antibiotic resistance Escherichia coli, this method is electrochemistry
Method, specifically includes the following steps:
Tested bacteria is placed in LB culture medium by step 3-1, stays overnight the bacterial culture growth；
The obtained bacterial cultures of step 3-1 is centrifuged by step 3-2；
Step 3-3 is washed the obtained bacterial cultures of step 3-2 three times with phosphate buffered saline solution, and will be remaining
Bacterial precipitation object is suspended in phosphate buffered saline solution for use；
Step 3-4 mixes the bacterial suspension that 3mL 1,4-benzoquinone solution and 3mL step 3-3 are obtained；
Step 3-5, after the E. coli suspension that step 3-4 is obtained incubates 1 hour, it is anti-that centrifugation terminates biocatalysis
It answers, and electrochemical analysis is carried out to sample supernatant.
Further, the concentration of 1,4-benzoquinone is 6mM in step 3-4.
Further, centrifugation rate is 3600rpm in step 3-5, and centrifugation time is 10 minutes.
Antibiotic resistance Escherichia coli preparation method disclosed by the invention is simple, can effectively induce with different
The Escherichia coli of resistance.
In antibiotic resistance Escherichia coli colorimetric detection method disclosed by the invention, can simply and rapidly be seen by naked eyes
It surveys and distinguishes Escherichia coli and other common clinical bacteriums, and it can be quantified.And another antibiotic resistance Escherichia coli electricity
Chemical measure can not only distinguish Escherichia coli and other common clinical bacteriums, moreover it is possible to potentially distinguish WT Escherichia coli and
Antibiotic resistance Escherichia coli.The equal simple and sensitive of two methods, convenient effective, low in cost, suitable for large-scale promotion and production,
With biggish application prospect.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is the minimum inhibitory concentration comparison diagram of the different methoxybenzyl aminopyrimidine resistant E. colis of artificial induction；
Fig. 2 is the oxidation peak disparity map of the 1,4-benzoquinone of various concentration；
Fig. 3 is different the color of e. coli concentration observation；
Fig. 4 is different e. coli concentration RBG value calibration curves；
Fig. 5 is the redox peak calibration function curve of different e. coli concentrations；
Fig. 6 is the redox peak figure of the Escherichia coli of different methoxybenzyl aminopyrimidine resistances；
Fig. 7 is the redox peak figure of the Escherichia coli of different Erythromycinresistants.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
In the accompanying drawings, the identical component of structure is indicated with same numbers label, everywhere the similar component of structure or function with
Like numeral label indicates.The size and thickness of each component shown in the drawings are to be arbitrarily shown, and there is no limit by the present invention
The size and thickness of each component.Apparent in order to make to illustrate, some places suitably exaggerate the thickness of component in attached drawing.It is all
The average value and standard deviation of concentration come from three repeated experiments.
Embodiment 1, microculture
Step 1, bacterium is placed in LB culture medium, stays overnight bacterial culture growth, cultivation temperature is 37 DEG C, oscillation
Frequency is 150rpm, and when culture is 12 hours a length of.
Step 2, the inoculum that step 1 obtains is centrifuged 10 minutes with the frequency of oscillation of 3600rpm and is delayed with phosphoric acid
Rush brine three times.Remaining bacterial precipitation object is suspended in phosphate buffered saline solution in case further using.
Embodiment 2, antibiotic-resistant bacteria induction
Antibiotic selection: one of Trimethoprim resistant, Erythromycinresistant.
Step 1, wild-type e. coli (WT) is inoculated into a series of conical flasks with dual antibiotic concentration gradient
In, final volume 100mL.Select the initial concentration of dual antibiotic concentration gradient for 50%MIC (minimum inhibitory concentration), it will
Each flask is 37 DEG C in temperature, and frequency of oscillation carries out oscillation bath under conditions of being 150rpm；
Step 2, after measuring process 2 each flask OD600It is worth (light absorption value of the solution at 600nm wavelength)；
Step 3, with the growth of bacterium, make the bacterial strain breeding with highest antibiotic concentration of bacterial growth to more
During another wheel antibiotic resistance of high antibiotic concentration gradient is incubated for；
Step 4, after 5 wheel selections, low antibiotic resistance Escherichia coli are collected；
Step 5, after 10 wheel selections, high antibiotic resistant E. coli is collected.
Low antibiotic Trimethoprim resistant Escherichia coli and low antibiotic erythromycin resistance large intestine bar can be obtained by step 4
Bacterium；High antibiotic Trimethoprim resistant Escherichia coli and high antibiotic Erythromycinresistant Escherichia coli are obtained by step 5.
The daily growth curve of both resistant E. colis and WT Escherichia coli is measured within every 6 hours, until Escherichia coli
Concentration is stablized.The MIC (minimum inhibitory concentration) of nonreactive Escherichia coli (WT) as shown in Figure 1 is 12.5 μ g/mL, and artificial induction's is low
The MIC of antibiotic Trimethoprim resistant Escherichia coli is that 120 μ g/mL, the MIC of high antibiotic Trimethoprim resistant Escherichia coli are
250 μ g/mL, compared with WT, the resistance to trimethoprim is about 10 times and 20 times.Illustrate to underwrite by the abductive approach and lure
Export the Escherichia coli with different resistances.
Embodiment 3, response of the various concentration 1,4-benzoquinone in electrochemical process
Step 1, be equipped with various concentration BQ (1,2,3,4,5,6,7,8,9 and 10mM) and Escherichia coli (1.0 ×
109CFU/mL)；
Step 2, BQ the and 3mL Escherichia coli (1.0 × 10 of the various concentration of 3mL in step 1 are taken9CFU/mL) in centrifuge tube
It is incubated for 1 hour.
Step 3, sample step 2 obtained was with 3600rpm centrifugation 10 minutes.The supernatant of sample is obtained for electrification
Learn detection.
Sample is monitored using traditional three-electrode system, the relation curve by drawing current signal and current potential indicates boundary
The CV curve in face obtains cyclic voltammetry (CV) curve, can be concluded that
1, with the increase of BQ concentration, the redox peaks of CV curve enhance.
2, under the conditions of the phosphate buffered saline solution of same volume, with the increase of BQ concentration, the redox of CV curve
Peak significantly rises.
3, BQ as shown in Figure 2 1mM to 6mM oxidation peak significant difference increase, after 6mM oxidation peak difference by
Gradually reduce.Therefore best BQ detectable concentration of the 6mM as Escherichia coli is selected.
Embodiment 4, colorimetric determination method
Select bacterium bacterial strain: WT Escherichia coli, Escherichia coli O 157: H7, enterococcus faecalis, streptococcus mutans, golden yellow Portugal
(low antibiotic Trimethoprim resistant is big for 4 kinds of antibiotic-resistant bacterias that grape coccus, S. pullonum and embodiment 2 obtain
Enterobacteria, low antibiotic erythromycin resistant E. coli, high antibiotic Trimethoprim resistant Escherichia coli, high antibiotic erythromycin
Resistant E. coli).
Step 1, tested bacteria is placed in LB culture medium, stays overnight the bacterial culture growth；
Step 2, bacterial cultures step 1 obtained is centrifuged；
Step 3, bacterial cultures in step 2 is washed three times with phosphate buffered saline solution, and by remaining bacterial precipitation
Object is suspended in phosphate buffered saline solution for use；
Step 4, difference obtained in 100 μ L 1,4-benzoquinone (6mM) and 100 μ L steps 3 is added in 96 hole microtest plates
(concentration is 1 × 10 to the bacterial suspension of concentration9CFU/mL,1×108CFU/mL,1×107CFU/mL,1×106CFU/mL,1×
105CFU/mL,1×104CFU/mL,1×103CFU/mL)；
Step 5, after reaction 1 hour, the color change of 96 orifice plates is recorded.
Analysis of test results:
(1) as shown in figure 3, it can be seen that apparent color reaction gradient under different e. coli concentrations.
(2) as shown in table 1, colorimetric detection method of the invention with detection WT Escherichia coli and Escherichia coli O 157: H7
Sample have color change, other kinds of bacterium (enterococcus faecalis, streptococcus mutans, staphylococcus aureus, white diarrhea sramana
Salmonella) it is unchanged to color, illustrate that this method being capable of the observable differentiation Escherichia coli of naked eyes and other common clinical bacteriums.
(3) observe by the naked eye or smart phone shooting image, then by RGB analyze number, by draw current signal and
The relation curve of current potential indicates the CV curve at interface.Difference e. coli concentration (10 as shown in Figure 43-109CFU/mL rgb value)
Calibration curve is matched with the function of y=742.16-48.75x, R2It is 0.96292.Error line represents duplicate measurements three times.Rgb value
It is red, the summation of green and blue value.It can be to 1.0 × 104The concentration of the Escherichia coli of CFU/mL or more is more accurately
It is quantitative.
Selective response of the colorimetric method of the present invention of table 1 to detection Escherichia coli
(3) as shown in table 2, (1.0 × 10 under identical concentration9CFU/mL) low antibiotic Trimethoprim resistant large intestine bar
The color of bacterium and low antibiotic erythromycin resistant E. coli is than without anticolibacillary of light color, and high antibiotic methoxy benzyl
The color of pyridine resistant E. coli and high antibiotic Erythromycinresistant Escherichia coli is more shallow.Illustrate colorimetric detection method of the invention
WT Escherichia coli and antibiotic resistance Escherichia coli can potentially be distinguished.
The colorimetric method of the present invention of table 2 is to different antibiotic resistants
(4) dilute identical Escherichia coli with tap water and phosphate buffered saline solution respectively, make concentration reach 1.0 ×
108CFU/mL), the color response and CV response curve measured with colorimetric method of the invention is essentially the same, can be ignored,
This shows that colorimetric method of the invention can be used for the test of water sample in reality.
Embodiment 5, electrochemical process detection method
Select bacterium bacterial strain: WT Escherichia coli, Escherichia coli O 157: H7, enterococcus faecalis, streptococcus mutans, golden yellow Portugal
(low antibiotic Trimethoprim resistant is big for 4 kinds of antibiotic-resistant bacterias that grape coccus, S. pullonum and embodiment 2 obtain
Enterobacteria, low antibiotic erythromycin resistant E. coli, high antibiotic Trimethoprim resistant Escherichia coli, high antibiotic erythromycin
Resistant E. coli).
Step 1, by tested bacteria in LB culture medium, stay overnight the bacterial culture growth；
Step 2, bacterial cultures step 1 obtained is centrifuged；
Step 3, it is washed three times with the bacterial cultures that phosphate buffered saline solution obtains step 2, and by remaining bacterium
Sediment is suspended in phosphate buffered saline solution for use；
Step 4, the bacterial suspension that mixing 3mL 1,4-benzoquinone solution and 3mL step 3 obtain；
Step 5, after bacterial suspension step 4 obtained incubates 1 hour, centrifugation terminates biocatalytic reaction, and to sample
It savors supernatant and carries out electrochemical analysis.
Analysis of test results:
(1) electrochemical detection method of the invention is 1 × 109WT Escherichia coli and large intestine bar are detected under CFU/mL concentration
The redox peaks of the sample of bacterium O157:H7 are remarkably decreased, and (enterococcus faecalis, streptococcus mutans are golden yellow for other kinds of bacterium
Color staphylococcus, S. pullonum) redox peaks it is unchanged, illustrate that this method can distinguish Escherichia coli and other
Common clinical bacterium.
(2) electrochemical detection method through the invention obtains cyclic voltammetry (CV) curve, as shown in figure 5, in large intestine
Bacillus concentration is 1.0 × 103To 1.0 × 109In the case where CFU/mL, redox peaks show that lining successively decreases, shown fitting function
For=0.00102-1.35297 × 10 IE.coli (μ A)-4× CE.coli (μ A), R2It is 0.99514.It can be detected by reading
1.0×103The concentration of the Escherichia coli of CFU/mL or more.
(3) as shown in fig. 6, electrochemical detection method through the invention measures (1.0 × 10 under identical concentration9CFU/
ML) wild-type e. coli (WT), low antibiotic Trimethoprim resistant Escherichia coli and high antibiotic Trimethoprim resistant large intestine
The redox peak of bacillus compares.As shown in fig. 7, electrochemical detection method through the invention measures under identical concentration
(1.0×109CFU/mL) wild-type e. coli (WT), low antibiotic erythromycin resistant E. coli and high antibiotic erythromycin
The redox peak of resistant E. coli compares.Electrochemical detection method of the invention of drawing a conclusion can potentially distinguish WT
Escherichia coli and antibiotic resistance Escherichia coli.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of antibiotic resistance Escherichia coli preparation method, which is characterized in that the preparation method comprises the following steps:
Wild-type e. coli (WT) is inoculated into a series of conical flasks with dual antibiotic concentration gradient by step 1-1
Middle culture, and carry out oscillation bath；
The OD of each conical flask after step 1-2, measuring process 1-1600It is worth (light absorption value of the solution at 600nm wavelength), with measurement
The concentration of wild-type e. coli (WT) culture solution；
Step 1-3, the bacterial strain breeding with highest antibiotic concentration for growing wild-type e. coli (WT) are higher to having
In the next round antibiotic resistance culture of antibiotic concentration gradient；
Step 1-4 collects low antibiotic resistance Escherichia coli after step 1-3 carries out 5 wheel selections；
Step 1-5 collects high antibiotic resistant E. coli after step 1-3 carries out 10 wheel selections.
2. preparation method according to claim 1, which is characterized in that dual antibiotic concentration gradient described in step 1-1
Initial concentration be minimum inhibitory concentration 50%.
3. preparation method according to claim 1, which is characterized in that the temperature for vibrating bath in step 1-1 is 37 DEG C, oscillation
Revolving speed is 150rpm.
4. a kind of detection method of the antibiotic resistance Escherichia coli mediated based on 1,4-benzoquinone, which is characterized in that the method is
Colorimetric method, specifically includes the following steps:
Tested bacteria is placed in LB culture medium by step 2-1, stays overnight the bacterial culture growth；
Step 2-2, the bacterial cultures that step 2-1 is obtained are centrifuged；
Step 2-3 is washed bacterial cultures in step 2-2 three times with phosphate buffered saline solution, and by remaining bacterial precipitation
Object is suspended in phosphate buffered saline solution for use；
Step 2-4 adds various concentration obtained in 100 μ L 1,4-benzoquinone and 100 μ L step 2-3 in 96 hole microtest plates
Bacterial suspension；
Step 2-5 records the color change of 96 orifice plates after reaction 1 hour.
5. detection method according to claim 4, which is characterized in that the growth time of bacterial cultures is in step 2-1
12 hours, temperature was 37 DEG C, and concussion revolving speed is 150rpm.
6. detection method according to claim 4, which is characterized in that centrifugation rate is 3600rpm, centrifugation in step 2-2
Time is 10 minutes.
7. detection method according to claim 4, which is characterized in that the concentration of 1,4-benzoquinone is 6mM in step 2-4, each thin
The concentration of bacterium suspension is respectively 1 × 109CFU/mL,1×108CFU/mL,1×107CFU/mL,1×106CFU/mL,1×
105CFU/mL,1×104CFU/mL,1×103CFU/mL。
8. a kind of detection method of antibiotic resistance Escherichia coli, which is characterized in that the method is electrochemical process, is specifically included
Following steps:
Tested bacteria is placed in LB culture medium by step 3-1, stays overnight the bacterial culture growth；
The obtained bacterial cultures of step 3-1 is centrifuged by step 3-2；
Step 3-3 is washed the obtained bacterial cultures of step 3-2 three times with phosphate buffered saline solution, and by remaining bacterium
Sediment is suspended in phosphate buffered saline solution for use；
Step 3-4 mixes the bacterial suspension that 3mL 1,4-benzoquinone solution and 3mL step 3-3 are obtained；
Step 3-5, after the E. coli suspension that step 3-4 is obtained incubates 1 hour, centrifugation terminates biocatalytic reaction, and
Electrochemical analysis is carried out to sample supernatant.
9. detection method according to claim 8, which is characterized in that the concentration of 1,4-benzoquinone is 6mM in step 3-4.
10. detection method according to claim 8, which is characterized in that centrifugation rate is 3600rpm, centrifugation in step 3-5
Time is 10 minutes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635926A (en) * | 2020-06-05 | 2020-09-08 | 上海交通大学 | Large-scale compound optimization method based on algorithm |
| CN112034021A (en) * | 2020-09-08 | 2020-12-04 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
| CN113122609A (en) * | 2021-04-27 | 2021-07-16 | 中国科学院长春应用化学研究所 | Simple and rapid colorimetric detection method for total toxicity of water body |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553954A (en) * | 2001-03-30 | 2004-12-08 | ������������ʳƷ�ۺ��о��� | Method for increasing productivity of secondary metabolite by conferring drug-resistant mutations |
| CN104267073A (en) * | 2014-08-11 | 2015-01-07 | 西安交通大学 | Method for detecting water pollutant biotoxicity by paper-based micro-fluidic chip anode current |
| CN108998499A (en) * | 2018-06-05 | 2018-12-14 | 上海海洋大学 | A kind of method of Escherichia coli Antibiotic Resistance in quick measurement biofilm |
-
2019
- 2019-04-08 CN CN201910276041.6A patent/CN110029082A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1553954A (en) * | 2001-03-30 | 2004-12-08 | ������������ʳƷ�ۺ��о��� | Method for increasing productivity of secondary metabolite by conferring drug-resistant mutations |
| CN104267073A (en) * | 2014-08-11 | 2015-01-07 | 西安交通大学 | Method for detecting water pollutant biotoxicity by paper-based micro-fluidic chip anode current |
| CN108998499A (en) * | 2018-06-05 | 2018-12-14 | 上海海洋大学 | A kind of method of Escherichia coli Antibiotic Resistance in quick measurement biofilm |
Non-Patent Citations (2)
| Title |
|---|
| 张智翔等: "组合抗性筛选法选育达托霉素高产菌株", 《中国抗生素杂志》 * |
| 王海霞等: "MPMS诱变结合抗生素抗性选育多杀菌素高产菌株", 《粮油食品科技》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111635926A (en) * | 2020-06-05 | 2020-09-08 | 上海交通大学 | Large-scale compound optimization method based on algorithm |
| CN112034021A (en) * | 2020-09-08 | 2020-12-04 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
| CN112034021B (en) * | 2020-09-08 | 2022-02-01 | 南京农业大学 | Method for detecting fluorine content in escherichia coli |
| CN113122609A (en) * | 2021-04-27 | 2021-07-16 | 中国科学院长春应用化学研究所 | Simple and rapid colorimetric detection method for total toxicity of water body |
| CN113122609B (en) * | 2021-04-27 | 2023-06-13 | 中国科学院长春应用化学研究所 | Simple and rapid colorimetric detection method for total toxicity of water body |
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