CN107656056A - A kind of method increased based on bacterium to the quick microscopy of bacterium - Google Patents
A kind of method increased based on bacterium to the quick microscopy of bacterium Download PDFInfo
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- CN107656056A CN107656056A CN201710757047.6A CN201710757047A CN107656056A CN 107656056 A CN107656056 A CN 107656056A CN 201710757047 A CN201710757047 A CN 201710757047A CN 107656056 A CN107656056 A CN 107656056A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000000386 microscopy Methods 0.000 title claims abstract description 26
- 230000001965 increasing effect Effects 0.000 title claims abstract description 10
- 241000588724 Escherichia coli Species 0.000 claims abstract description 36
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims abstract description 30
- 239000012528 membrane Substances 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 11
- 230000003115 biocidal effect Effects 0.000 claims abstract description 8
- 150000003952 β-lactams Chemical class 0.000 claims abstract 2
- 230000004048 modification Effects 0.000 claims description 36
- 238000012986 modification Methods 0.000 claims description 36
- 239000003782 beta lactam antibiotic agent Substances 0.000 claims description 33
- 239000002132 β-lactam antibiotic Substances 0.000 claims description 33
- 229940124586 β-lactam antibiotics Drugs 0.000 claims description 33
- WZPBZJONDBGPKJ-VEHQQRBSSA-N aztreonam Chemical compound O=C1N(S([O-])(=O)=O)[C@@H](C)[C@@H]1NC(=O)C(=N/OC(C)(C)C(O)=O)\C1=CSC([NH3+])=N1 WZPBZJONDBGPKJ-VEHQQRBSSA-N 0.000 claims description 25
- 230000002401 inhibitory effect Effects 0.000 claims description 16
- 238000012545 processing Methods 0.000 claims description 15
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- 229940106164 cephalexin Drugs 0.000 claims description 10
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- 229960003022 amoxicillin Drugs 0.000 claims description 8
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 3
- 241000305071 Enterobacterales Species 0.000 description 3
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- 241001646719 Escherichia coli O157:H7 Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical class ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 108700020474 Penicillin-Binding Proteins Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention is designed and establishes a kind of new method for being used for microscopy detection Escherichia coli and salmonella typhimurium increased based on bacterium.Increased by adding beta-lactam class antibiotic microorganism into testing sample solution, and combined membrane filtering realizes the specificity and high-sensitivity detection to Escherichia coli and salmonella typhimurium with Magneto separate, so that number of bacteria can be judged under an optical microscope, this method may apply in water sample, and whole detection process can be completed in 2 hours.Experiment proves that the inventive method to the test limit of Escherichia coli and two kinds of bacteriums of salmonella typhimurium can as little as 5 cells, the requirement of quick determination method can be met.This method is simple to operate, and cost is low, can be widely applied in actual sample in the microscopy detection of Gram-negative bacteria.
Description
Technical field
It is more particularly to a kind of to be increased based on bacterium to the quick microscopy of bacterium the present invention relates to technical field of microbial detection
Method.
Background technology
The food pollution as caused by microorganism and food origin disease have greatly threatened the health and food service industry of the public
Development.In worldwide, the disease triggered every year by food-borne pathogens such as Escherichia coli and salmonella endangers pole
To be serious.Even very low infective dose can all cause serious disease.Such as the infective dose of Escherichia coli and salmonella
Can as little as 10 cells.Therefore the number for establishing effective detection method detection bacterium is particularly important.
Traditional flat board cultural method is the national standard method of current Bacteria Detection, but this method is cumbersome, consumption
When it is longer, the requirement of quick detection can not be met.EUSA, PCR and surface-enhanced Raman
The detection methods such as spectrum have high sensitivity, and quickly, the advantages that high specificity, but instrumentation is more complicated, spends cost ratio
It is higher, it is necessary to complicated sample handling processes.It is not suitable for Site Detection.And when cell quantity is less than 100, quick detection
The degree of accuracy of method is not high.Micro- sem observation is to compare intuitively method, can visually judge the number of bacterium.This method is grasped
Make simply, cost expenses are less, are not required to the sample handling processes of complexity.But length of bacteria is generally less than 5 μm, it is difficult to clearly
Itself and impurity are differentiated.Therefore establish it is a kind of based on microscopical detection method to realize on individual cell level to the fast of bacterium
Speed detection turns into this area urgent problem to be solved.
The content of the invention
Beta-lactam antibiotic can be by suppressing or inducing PBP (penicillin binding
Proteins, PBPs) mutation is produced, the polymerization of peptide glycan in cell membrane is influenceed, makes bacterial cell wall defect, thalline expansion is split
Solution.Based on this, inventor has found under study for action, passes through the proper treatment of antibiotic, the fission process of Gram-negative bacteria
It is suppressed, but cell length can increase in thread until cell dissolves always, and in the process, bacterium after growth can be with
Under the microscope it can be clearly seen that.Based on this, inventor proposes a kind of side for increasing based on bacterium and realizing to the quick microscopy of bacterium
Method, this method is simple to operate, takes short, and sensitivity is high, and specificity is good, it is not necessary to costly equipment, cost is cheap, suitable for pair
The practical application of bacterioscopy.
Specifically, the present invention relates to following technical scheme：
The first aspect of the invention, disclose application of the beta-lactam antibiotic in the quick microscopy of bacterium.
Wherein, beta-lactam antibiotic includes but is not limited to penicillin and its derivative, cynnematin, monoamides ring
Class, Carbapenems and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, and ammonia is bent
South and Amoxicillin；Still more preferably, the beta-lactam antibiotic is AZT, and the AZT addition concentration is
1~100 times of minimal inhibitory concentration (being preferably 1 times of minimal inhibitory concentration)；Experiment shows that AZT is in 1 times of minimal inhibitory concentration
When Escherichia coli and salmonella typhimurium thalline can be caused to increase to the full extent, without causing thalline to be measured quickly to dissolve,
Be advantageous to follow-up microscopy；
The bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
It is described application specific method be：Beta-lactam antibiotic is added in tested bacteria, promotes microorganism to stretch
It is long, it is easy to microscopy observation under the microscope.
Further, the application specific method is：By AZT with final concentration of 1 times of minimal inhibitory concentration added to greatly
In enterobacteria or salmonella typhimurium bacterium solution.
The second aspect of the invention, disclose a kind of method increased based on bacterium to the quick microscopy of bacterium, specific bag
It is as follows to include step：
S1. add beta-lactam antibiotic into testing sample and antibody modification magnetic ball carries out incubation processing, wherein, β-
Lactam antibiotics promotes tested bacteria thalline to extend, and is easy to follow-up microscopy under the microscope to observe；Antibody modification magnetic ball with
Bacterium coupling after elongation；
S2. the testing sample after step S1. processing is subjected to membrane filtration, captured bacterium can pass through membrane filtration operation
It is trapped on filter membrane, filter membrane then is carried out into Magneto separate processing further goes the removal of impurity；
S3. the filter membrane after step S2. processing is placed in micro- Microscopic observation and counted.
Wherein, bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
Beta-lactam antibiotic includes but is not limited to penicillin and its derivative, cynnematin, monoamides ring class, carbon are blue or green
Mould alkenes and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, AZT and A Mo
XiLin；Still more preferably, the beta-lactam antibiotic is AZT；
Antibody modification magnetic ball preparation is：
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature bar
Activated under part, then carry out Magneto separate, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
2) corresponding bacterial antibodies are added into the magnetic bead after activation, are reacted under room temperature condition, Magneto separate is then molten with PBS
Liquid is cleaned repeatedly, and then the magnetic ball of antibody modification is added in PBS-BSA solution and closed, and concussion processing, then uses PBS solution
Antibody modification magnetic ball is resuspended to obtain.
Wherein, antibody is anti-Escherichia coli antibody or anti-salmonella typhimurium antibody, and the antibody addition concentration is 0.1
~5 μ g/mL (being preferably 2 μ g/mL)；
The third aspect of the invention, application of the above-mentioned detection method in quick bacteria microscopy is disclosed, specifically, institute
It is to carry out quick microscopy to Escherichia coli in drinking water and/or salmonella typhimurium to state application mode.
Beneficial effects of the present invention：
The present invention, which designs and establishes a kind of microscopy that is used for based on bacterium growth, detects Escherichia coli and mouse typhus sramana
The new method of Salmonella.Extended by adding beta-lactam antibiotic microorganism into testing sample solution, joined simultaneously
Close specificity and high-sensitivity detection of the membrane filtration with Magneto separate realization to Escherichia coli and salmonella so that number of bacteria exists
It can be judged under light microscope, this method may apply in water sample, and whole detection process can be completed in 2 hours.
Experiment proves that the inventive method can be as little as 5 thin to the test limit of Escherichia coli and two kinds of bacteriums of salmonella typhimurium
Born of the same parents, the requirement of quick determination method can be met.This method is simple to operate, and cost is low, high sensitivity, and specificity is good, can be extensive
Applied in actual sample in the detection of the microscopy of Gram-negative bacteria.
Brief description of the drawings
Figure 1A is that the bacterium after increasing is coupled schematic diagram with antibody modification magnetic ball；Figure 1B is the present invention based on bacterium growth pair
The quick microscopy method flow schematic diagram of bacterium；
Fig. 2A is various concentrations antibiotic to coli somatic growth pattern；Fig. 2 B are that various concentrations antibiotic is hindered to mouse
Cold salmonella thalline growth pattern；In figure, AMX represents A Mosilin；ATM represents AZT, and CEL represents cefalexin, MIC
Represent minimum inhibitory concentration；
Fig. 3 A are magnetic ball microscope figure；Fig. 3 B are the magnetic ball after Escherichia coli (a) are closed with salmonella typhimurium (c)
Plated growth situation after capture.Escherichia coli (b) are with salmonella typhimurium (d) by flat after the magnetic ball capture of antibody modification
Plate growing state；Fig. 3 C are the fluorescence intensity of the magnetic ball compound of surface modification various concentrations fluorescence antibody；
Fig. 4 is that Escherichia coli (a) are shown with salmonella typhimurium (c) after the magnetic ball capture of antibody modification on wave carrier piece
Diagram；Escherichia coli (b) are shown figure after the magnetic ball capture of antibody modification with salmonella typhimurium (d) on filter membrane；
Fig. 5 a are that the inventive method investigates block diagram to Escherichia coli specificity, and Fig. 5 b are that the inventive method is husky to mouse typhus
Door Salmonella specificity investigates block diagram.
Embodiment
It is noted that described further below is all exemplary, it is intended to provides further instruction to the application.It is unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
As described in background technology, it is low detection sensitivity to be present in food-borne pathogens detection method, detection process
Cumbersome or detection process need to use the defects of Large expensive instrument.
In view of this, in a kind of embodiment of the invention, there is provided beta-lactam antibiotic is in the quick mirror of bacterium
Application in inspection.
Wherein, beta-lactam antibiotic includes but is not limited to penicillin and its derivative, cynnematin, monoamides ring
Class, Carbapenems and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, and ammonia is bent
South and Amoxicillin；Still more preferably, the beta-lactam antibiotic is AZT.
The bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
In the still another embodiment of the present invention, the application specific method is：Beta-lactam antibiotic is added
Into tested bacteria, promote microorganism to extend, be easy to microscopy observation under the microscope.
Further, the application specific method is：By AZT with final concentration of 1 times of minimal inhibitory concentration added to greatly
In enterobacteria or salmonella typhimurium bacterium solution.
In the still another embodiment of the present invention, there is provided a kind of method increased based on bacterium to the quick microscopy of bacterium,
It is as follows to specifically include step：
S1. add beta-lactam antibiotic into testing sample and antibody modification magnetic ball carries out incubation processing, wherein, β-
Lactam antibiotics promotes tested bacteria thalline to extend, and is easy to follow-up microscopy under the microscope to observe；Antibody modification magnetic ball with
Bacterium coupling after elongation；
S2. the testing sample after step S1. processing is subjected to membrane filtration, captured bacterium can pass through membrane filtration operation
It is trapped on filter membrane, filter membrane then is carried out into Magneto separate processing further goes the removal of impurity；
S3. the filter membrane after step S2. processing is placed in micro- Microscopic observation and counted.
Wherein, bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
Beta-lactam antibiotic includes but is not limited to penicillin and its derivative, cynnematin, monoamides ring class, carbon are blue or green
Mould alkenes and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, AZT and A Mo
XiLin；Still more preferably, the beta-lactam antibiotic is AZT；The AZT addition concentration is 1~100 times
Minimal inhibitory concentration (being preferably 1 times of minimal inhibitory concentration)；
Antibody modification magnetic ball preparation is：
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature bar
Activated under part, then carry out Magneto separate, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
2) corresponding bacterial antibodies are added into the magnetic bead after activation, are reacted under room temperature condition, Magneto separate is then molten with PBS
Liquid is cleaned repeatedly, and then the magnetic ball of antibody modification is added in PBS-BSA solution and closed, and concussion processing, then uses PBS solution
Antibody modification magnetic ball is resuspended to obtain.
Wherein, antibody is anti-Escherichia coli antibody or anti-salmonella typhimurium antibody, and the antibody addition concentration is 0.1
~5 μ g/mL (being preferably 2 μ g/mL)；
In the still another embodiment of the present invention, application of the above-mentioned detection method in quick bacteria microscopy is disclosed,
Specifically, the application mode is to carry out quick microscopy to Escherichia coli in drinking water and/or salmonella typhimurium.
Mode by the following examples is further elaborated to the present invention.
Embodiment
1. materials and methods
Escherichia coli O 157:H7 (ATCC 43888) is bought in China General Microbiological with salmonella (ATCC 14028)
Culture presevation administrative center.Anti- Escherichia coli antibody, anti-salmonella antibody are bought with marked by fluorescein isothiocyanate IgG antibody
In Beijing Bo Aosen Bioisystech Co., Ltd.Aminopolystyrene magnetic ball (3-4 μm of 1mg/ml, carboxyl magnetic ball) is bought in the U.S.
Aladdin industrial group.Polycarbonate membrane (5 μm) is bought in Shanghai Fu Xue bio tech ltd.Carbodiimides hydrochloric acid
Salt, N- hydroxysuccinimides are bought in upper sea green source bio tech ltd.Cefalexin, AZT, Amoxicillin purchase
Buy and hold gloomy biological Co., Ltd in Jinan.Beef extract, peptone, yeast extract, agar powder are bought in the extensive and profound in meaning star biology skill in Beijing
Art Co., Ltd.
2. related solution is prepared
EDC/NHS solution is prepared：EDC 9.6mg is weighed respectively, NHS 9.6mg is dissolved in 3mL secondary waters.
0.01mol·L-1Phosphate buffer solution is prepared：Na is weighed respectively2HPO4·12H2O 13.76g、NaH2PO4·
2H2O 1.79g, NaCl 9.00g, add deionized water to be settled to 1000mL.
SYBR Green Ι standard solutions are prepared：SYBR Green Ι stostes are diluted 100 times with DMSO, -20 DEG C of preservations.
EDTA-2Na is prepared using liquid：EDTA-2Na 18.61g are weighed, add sterile purified water to be settled to 100mL.
LB fluid nutrient mediums are prepared：Tryptone 10g, yeast extract 5g, NaCl 10g are weighed respectively, are determined with deionized water
Hold to 1000mL, adjust pH to 7.0.
Beef-protein medium is prepared：Beef extract 3g, peptone 10g, NaCl 5g are weighed respectively, use deionized water
1000mL is settled to, adds 20g agar.
3. Bacteria Culture and sample treatment
With oese, picking Escherichia coli are fallen within LB fluid nutrient mediums with salmonella typhimurium single bacterium from inclined-plane, and 37
DEG C shaken cultivation 12h.After bacteria suspension is diluted into 1000 times with fresh LB, 37 DEG C of shaken cultivation 2h.Cefalexin, ammonia are bent
South, Amoxicillin are diluted to 1,10,100 times of minimal inhibitory concentration, are added in bacteria suspension respectively, 37 DEG C of culture 2h.Every
0.5h observes Bacteria cold shock using inverted microscope and photographed to record.The length change of bacterium passes through Nano Measurer
Software is calculated.
4. the preparation of antibody modification magnetic ball
200 μ L carboxyl magnetic ball ultrasound 30s are taken, Magneto separate, are cleaned once with distilled water.250 μ L EDC/NHS solution are added,
30min is activated at ambient temperature, Magneto separate, is cleaned repeatedly with PBS solution 3 times, produces magnetic ball after activation；
The anti-Escherichia coli antibody of 150 μ L and anti-salmonella typhimurium antibody, room temperature are separately added into magnetic ball after activation
Under the conditions of react 6h, Magneto separate, use 0.01molL-1PBS solution is cleaned 3 times repeatedly.The magnetic ball of antibody modification is added to 1%
Closed in BSA-PBS solution, 37 DEG C shake 1h.Then 0.01molL is used-1The magnetic ball of antibody modification is resuspended PBS 1mL,
It is placed in 4 DEG C.
5. the fluorescent staining method of bacterium
1mL bacterium solutions are taken in polystyrene sample pipe, 5min is preheated in 37 DEG C of water-baths, adding 10 μ L EDTA-2Na makes
With liquid, 10 μ L SYBR Green Ι standard solutions are rapidly added after mixing, 37 DEG C of lucifuges are incubated 10min, 4 DEG C of 3000r
min-15min is centrifuged, abandons supernatant (removing culture medium and other impurities), PBS is centrifuged again after redissolving.Above-mentioned centrifugally operated is repeated, is abandoned
Supernatant, it is stand-by that PBS redissolves precipitation.
6. the capture and detection of bacterium
The magnetic ball of antibody modification and beta-lactam antibiotic are added in bacteria suspension simultaneously, 37 DEG C of incubation 1.5h.Increase
Bacterium after length can be by the magnetic ball specificity capture of antibody modification.Captured bacterium can be trapped by membrane filtration operation
Onto filter membrane, filter membrane is put into can further remove the impurity being trapped on filter membrane with PBS on Magneto separate frame.Then will
Filter membrane is placed in micro- Microscopic observation and counted.
As a result with discussion
1. the optimization of bacterium condition of growth
Beta-lactam antibiotic includes penicillin, monobactams of cynnematin and new development etc..Cephalo
Ammonia benzyl, AZT and Amoxicillin are representative substances.Three kinds of antibiotic are added separately in bacterium solution to final concentration of 1,10,
100 times of minimal inhibitory concentrations.37 DEG C of shaken cultivation 2h.As shown in Fig. 2 compared to cefalexin, Amoxicillin, AZT concentration
For 1 times of minimal inhibitory concentration (128 μ gmL-1) when Escherichia coli (A) and salmonella (B) thalline can be caused to increase to the full extent
It is long.When three kinds of antibiotic concentrations are 100 times of minimal inhibitory concentrations, in one hour dissolution phenomena occurs for bacterium.Because mostly
The typical beta-lactam antibiotic of number can all cause cell to dissolve in high concentration.Therefore, Antibiotics and concentration optimization
For AZT, 1 times of minimal inhibitory concentration.
2. the sign of magnetic ball
Magnetic ball is characterized by microphotograph.As shown in Figure 3A, in PBS favorable dispersibility and ball is presented in magnetic ball
Shape state.The magnetic ball of antibody modification is added in bacterium solution and is incubated simultaneously with AZT.Bacterium can be special by the magnetic ball of antibody modification
Property capture.Magnetic ball is separated by Magneto separate frame, then using 0.01molL-1PBS is flushed three times.Magnetic ball is resuspended in 100 μ
L0.01mol·L-1In PBS, on even spread to beef extract-peptone flat board.37 DEG C of culture 24h.Magnetic ball after closing is added to
In bacterium solution as a control group.As shown in Figure 3 B, the Escherichia coli (a) and salmonella typhimurium that the magnetic ball after closing is captured
(c) do not grown on flat board, illustrate that the magnetic ball after closing does not have non-specific adsorption to bacterium.Caught by the magnetic ball of antibody modification
The Escherichia coli (b) obtained and salmonella typhimurium (d) well-grown on flat board, show antibody modification magnetic ball can with it is thin
Bacterium successfully combines.
3. the concentration optimization of magnetic ball surface modified antibodies
The IgG antibody of FITC marks arrives magnetic ball surface as model modification.The concentration of fixed magnetic ball, various concentrations
Magnetic ball surface is arrived in FITC-IgG modifications, and its fluorescence intensity is detected with XRF.As shown in Figure 3 C, when FITC-IgG concentration from
0.1μg·mL-1Increase to 2.0 μ gmL-1When, the fluorescence intensity of compound gradually strengthens.When FITC-IgG concentration is 2.0 μ g/
During mL, complex fluorescence intensity nearly reaches maximum.And with the increase of FITC-IgG concentration, the fluorescence intensity of compound
Change unobvious, and as shown in Figure 3 C, when FITC-IgG antibody concentrations are 2.0 μ g/mL, fluorescence intensity is apparently higher than control group
(0 μ g/mL), show that antibody is coupled successfully with magnetic ball, therefore it is 2.0 μ g/mL to select optimization antibody concentration.
4. the detection of Escherichia coli and salmonella in water sample
Escherichia coli are added in water sample to examine the feasibility of this method with salmonella.It is 5,10,50 by quantity,
100 bacterium is added in water, then by LB culture mediums and water sample with 9:1 volume mixture.By AZT and antibody modification
Magnetic ball is added in water sample simultaneously, 37 DEG C of incubation 1.5h.By membrane filtration joint Magneto separate operation, filter membrane is put under microscope
Observation.The Escherichia coli (a) that Fig. 4 represents to be captured by magnetic ball are with salmonella typhimurium (c) on slide.Bacterium after growth
There is bigger surface area, can be closed with more magnetic chous, so as to improve the capture rate of magnetic ball.Fig. 4 (b, d) is respectively by magnetic
The Escherichia coli of ball capture are trapped on filter membrane with salmonella typhimurium after membrane filtration and Magneto separate operation.Table 1 is big
Enterobacteria (a) is added to the rate of recovery in water sample with salmonella typhimurium (b).As shown in the result of table 1, when the number of addition cell
Measure for 50-100 when, the rate of recovery is 92.00 ± 7.94%-94.67 ± 8.96%, when cell addition is 5-10, large intestine bar
The rate of recovery of bacterium and salmonella be respectively 106.67 ± 4.16%-83.33 ± 4.93%, 113.33 ± 2.52%-86.67 ±
3.70%.The test limit of two kinds of bacteriums can as little as 5 cells, the requirement of quick determination method can be met.
Table 1
5. the inventive method specific assay
Escherichia coli, after salmonella typhimurium is dyed with staphylococcus aureus with SYBR Green I, while add anti-
The magnetic ball and AZT of body modification.Surface modification has anti-Escherichia coli antibody and the magnetic ball of anti-salmonella typhimurium antibody to distinguish
It is added in bacterium solution, 37 DEG C of incubation 1.5h.Magneto separate and membrane filtration operation are carried out respectively, it is thin on film being trapped into PBS
Bacterium elutes fluorescence intensity.As shown in Figure 5 a, after adding the magnetic ball of anti-Escherichia coli antibody modification, Escherichia coli it is glimmering
Luminous intensity is apparently higher than salmonella typhimurium and staphylococcus aureus.Fig. 5 b represent to add anti-salmonella typhimurium antibody
Afterwards, Escherichia coli can be ignored with the fluorescence intensity of staphylococcus aureus compared with salmonella typhimurium.The experiment
Show the non-specific adsorption very little of magnetic ball and this method has and compares high specific and selectivity.Due to beta-lactam antibiotic not
The growth of gram-positive bacteria can be caused, be applied to detect Gram-negative bacteria in this way.
The preferred embodiment of the application is the foregoing is only, is not limited to the application, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. application of the beta-lactam antibiotic in the quick microscopy of bacterium.
2. application as claimed in claim 1, it is characterised in that the beta-lactam antibiotic includes penicillin and its derivative
Thing, cynnematin, monoamides ring class, Carbapenems and penems enzyme inhibitor.
Preferably, the beta-lactam antibiotic includes cefalexin, AZT and Amoxicillin (being most preferably AZT).
3. application as claimed in claim 1, it is characterised in that the bacterium is Gram-negative bacteria, it is preferred that the bacterium is
Escherichia coli and/or salmonella typhimurium.
4. application as claimed in claim 1, the application specific method are：Beta-lactam antibiotic is added to tested bacteria
In；
Preferably, the application specific method is：AZT is added to Escherichia coli with final concentration of 1 times of minimal inhibitory concentration
Or in salmonella typhimurium bacterium solution.
A kind of 5. method increased based on bacterium to the quick microscopy of bacterium, it is characterised in that as follows including step：
S1. add beta-lactam antibiotic into testing sample and antibody modification magnetic ball carries out incubation processing, wherein, β-interior acyl
Amine antibiotic promotes tested bacteria thalline to extend, and is easy to follow-up microscopy under the microscope to observe；Antibody modification magnetic ball and elongation
Bacterium coupling afterwards；
S2. the testing sample after step S1. processing is subjected to membrane filtration, captured bacterium can be cut by membrane filtration operation
It is left on filter membrane, filter membrane then is carried out into Magneto separate processing further goes the removal of impurity；
S3. the filter membrane after step S2. processing is placed in micro- Microscopic observation and counted.
6. method as claimed in claim 5, it is characterised in that the bacterium is Gram-negative bacteria, it is preferred that the bacterium is
Escherichia coli and/or salmonella typhimurium.
7. method as claimed in claim 5, it is characterised in that the beta-lactam antibiotic includes penicillin and its derivative
Thing, cynnematin, monoamides ring class, Carbapenems and penems enzyme inhibitor, it is preferred that the beta-lactam antibiosis
Element includes cefalexin, AZT and Amoxicillin；Still more preferably, the beta-lactam antibiotic is AZT,
The AZT addition concentration is 1~100 times of minimal inhibitory concentration (being preferably 1 times of minimal inhibitory concentration).
8. method as claimed in claim 5, it is characterised in that antibody modification magnetic ball preparation method is：
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate, is cleaned with distilled water；EDC/NHS solution is added thereto, at ambient temperature
Activation, then Magneto separate is carried out, magnetic bead after must being activated after being cleaned repeatedly with PBS solution；
2) corresponding bacterial antibodies are added into the magnetic bead after activation, are reacted under room temperature condition, Magneto separate is then anti-with PBS solution
The magnetic ball of antibody modification, is then added in PBS-BSA solution and closes by multiple cleaning, concussion processing, is then resuspended with PBS solution
Obtain antibody modification magnetic ball.
9. method as claimed in claim 8, it is characterised in that the antibody is anti-Escherichia coli antibody or anti-Salmonella typhimurium
Bacteria antibody, the antibody addition concentration are 0.1~5 μ g/mL (being preferably 2 μ g/mL).
10. application of any one of the claim 5-9 methods described in quick bacteria microscopy, it is preferred that the application mode is
Quick microscopy is carried out to Escherichia coli in drinking water and/or salmonella typhimurium.
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