CN107656056B - A method of increased based on bacterium to the quick microscopy of bacterium - Google Patents
A method of increased based on bacterium to the quick microscopy of bacterium Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000000386 microscopy Methods 0.000 title claims abstract description 25
- 230000001965 increasing effect Effects 0.000 title claims abstract description 10
- 241000588724 Escherichia coli Species 0.000 claims abstract description 34
- 239000003782 beta lactam antibiotic agent Substances 0.000 claims abstract description 30
- 239000002132 β-lactam antibiotic Substances 0.000 claims abstract description 30
- 229940124586 β-lactam antibiotics Drugs 0.000 claims abstract description 30
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 claims abstract description 27
- 239000012528 membrane Substances 0.000 claims abstract description 18
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- 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 21
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
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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
Abstract
The present invention is designed and establishes a kind of new method for microscopy detection Escherichia coli and salmonella typhimurium increased based on bacterium.By adding beta-lactam antibiotic into testing sample solution microorganism is increased, and combined membrane filtering and Magneto separate are realized and are detected to the specificity of Escherichia coli and salmonella typhimurium with highly sensitive, so that judgement can be obtained in number of bacteria under an optical microscope, this method can be applied in water sample, and entire detection process can be completed in 2 hours.Experiment proves that the method for the present invention can satisfy the requirement of rapid detection method down to 5 cells to the detection limit of Escherichia coli and two kinds of bacteriums of salmonella typhimurium.This method is easy to operate, at low cost, can be widely applied in actual sample in the microscopy detection of Gram-negative bacteria.
Description
Technical field
It is the present invention relates to technical field of microbial detection, in particular to a kind of to be increased based on bacterium to the quick microscopy of bacterium
Method.
Background technique
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, pole is endangered by the disease that food-borne pathogens such as Escherichia coli and salmonella are caused every year
It is serious.Even very low infective dose can all lead to serious disease.Such as the infective dose of Escherichia coli and salmonella
It can be down to 10 cells.Therefore the number for establishing effective detection method detection bacterium is particularly important.
Traditional plate cultural method is the national standard method of current Bacteria Detection, but this method is cumbersome, consumption
When it is longer, be unable to satisfy the requirement quickly detected.Enzyme-linked immunosorbent assay, polymerase chain reaction and surface-enhanced Raman
The detection methods such as spectrum have high sensitivity, and quickly, the advantages that high specificity, but instrumentation is more complex, spend cost ratio
It is higher, need complicated sample handling processes.It is not suitable for on-site test.And when cell quantity is lower than 100, quickly detect
The accuracy of method is not high.Micro- sem observation is the intuitive method of comparison, can visually judge the number of bacterium.This method behaviour
Make simply, cost expenses are less, are not required to complicated sample handling processes.But length of bacteria is generally less than 5 μm, it is difficult to clearly
It is differentiated with impurity.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 becomes this field urgent problem to be solved.
Summary of the invention
Beta-lactam antibiotic can be by inhibiting or inducing penicillin binding protein (penicillin binding
Proteins, PBPs) mutation is generated, the polymerization of peptide glycan in cell wall is influenced, bacterial cell wall defect is made, thallus 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 always in filiform until cell dissolution, 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 easy to operate, time-consuming short, and sensitivity is high, and specificity is good, does not need costly equipment, low in cost, is suitable for pair
The practical application of bacterioscopy.
Specifically, the present invention relates to following technical schemes:
The first aspect of the invention discloses 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, cephalosporin, 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 aztreonam, and the aztreonam addition concentration is
1~100 times of minimal inhibitory concentration (preferably 1 times of minimal inhibitory concentration)；Experiments have shown that aztreonam is in 1 times of minimal inhibitory concentration
When Escherichia coli and salmonella typhimurium thallus can be caused to increase to the full extent, without causing thallus to be measured quickly to dissolve,
Be conducive to subsequent microscopy；
The bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
The application method particularly includes: beta-lactam antibiotic is added in tested bacteria, microorganism is promoted to stretch
It is long, convenient for the observation of microscopy under the microscope.
Further, the application method particularly includes: be added to aztreonam greatly with final concentration of 1 times of minimal inhibitory concentration
In enterobacteria or salmonella typhimurium bacterium solution.
The second aspect of the invention discloses a kind of method increased based on bacterium to the quick microscopy of bacterium, specific to wrap
Include that steps are as follows:
S1. beta-lactam antibiotic is added into sample to be tested and antibody modification magnetic ball carries out incubation processing, wherein β-
Lactam antibiotics promote tested bacteria thallus to extend, and observe convenient for subsequent microscopy under the microscope；Antibody modification magnetic ball with
Bacterium coupling after elongation；
S2. step S1. treated sample to be tested is subjected to film filtering, captured bacterium can pass through membrane filtration operation
It is trapped on filter membrane, filter membrane is then subjected to Magneto separate processing further removal impurity；
S3. by step S2., treated that filter membrane is placed in microscopically observation and counts.
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 that penicillin and its derivative, cephalosporin, monoamides ring class, carbon are green
Mould alkenes and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, aztreonam and A Mo
XiLin；Still more preferably, the beta-lactam antibiotic is aztreonam；
Antibody modification magnetic ball preparation are as follows:
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature item
It is activated under part, then carries 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, react under room temperature, Magneto separate is then molten with PBS
Liquid cleans repeatedly, and then the magnetic ball of antibody modification is added in PBS-BSA solution and is closed, then PBS solution is used in concussion processing
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 (preferably 2 μ g/mL)；
The third aspect of the invention discloses application of the above-mentioned detection method in quick bacteria microscopy, specifically, institute
Stating application mode is to carry out quick microscopy to Escherichia coli in drinking water and/or salmonella typhimurium.
Beneficial effects of the present invention:
The present invention design and establish it is a kind of based on bacterium increase for microscopy detection Escherichia coli and mouse typhus sramana
The new method of Salmonella.By adding beta-lactam antibiotic into testing sample solution microorganism is extended, joins simultaneously
It closes film filtering to detect with specificity of the Magneto separate realization to Escherichia coli and salmonella with highly sensitive, so that number of bacteria exists
Judgement can be obtained under optical microscopy, this method can be applied in water sample, and entire detection process can be completed in 2 hours.
Experiment proves that the method for the present invention can be thin down to 5 to the detection limit of Escherichia coli and two kinds of bacteriums of salmonella typhimurium
Born of the same parents can satisfy the requirement of rapid detection method.This method is easy to operate, at low cost, high sensitivity, and specificity is good, can be extensive
Applied in actual sample in the detection of the microscopy of Gram-negative bacteria.
Detailed description of the invention
Figure 1A is that the bacterium and antibody modification magnetic ball after increasing are coupled schematic diagram；Figure 1B is that the present invention is based on bacterium growths pair
The quick microscopy method flow schematic diagram of bacterium；
Fig. 2A is various concentration antibiotic to coli somatic growth pattern；Fig. 2 B is that various concentration antibiotic hurts mouse
Cold salmonella thallus growth pattern；In figure, AMX represents A Mosilin；ATM represents aztreonam, and CEL represents cefalexin, MIC
Represent minimum inhibitory concentration；
Fig. 3 A is magnetic ball microscope figure；Fig. 3 B is the magnetic ball after Escherichia coli (a) are closed with salmonella typhimurium (c)
Plated growth situation after capture.Escherichia coli (b) and salmonella typhimurium (d) are by flat after the magnetic ball capture of antibody modification
Plate growing state；Fig. 3 C is the fluorescence intensity of the magnetic ball compound of surface modification various concentration fluorescence antibody；
Fig. 4 is that Escherichia coli (a) and salmonella typhimurium (c) are shown on wave carrier piece after the magnetic ball capture of antibody modification
Diagram；Escherichia coli (b) and salmonella typhimurium (d) are shown figure after the magnetic ball capture of antibody modification on filter membrane；
Fig. 5 a is that the method for the present invention investigates histogram to Escherichia coli specificity, and Fig. 5 b is the method for the present invention to mouse typhus sand
Door Salmonella specificity investigates histogram.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has 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 specific 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 singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
As described in background technique, food-borne pathogens detection method there are detection sensitivities low, detection process
Cumbersome or detection process need to use the defects of Large expensive instrument.
In view of this, providing beta-lactam antibiotic in the quick mirror of bacterium in a kind of specific embodiment of the invention
Application in inspection.
Wherein, beta-lactam antibiotic includes but is not limited to penicillin and its derivative, cephalosporin, 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 aztreonam.
The bacterium is Gram-negative bacteria, and further, the bacterium is Escherichia coli and/or Salmonella typhimurium
Bacterium；
In still another embodiment of the invention, the application method particularly includes: add beta-lactam antibiotic
Into tested bacteria, microorganism is promoted to extend, convenient for the observation of microscopy under the microscope.
Further, the application method particularly includes: be added to aztreonam greatly with final concentration of 1 times of minimal inhibitory concentration
In enterobacteria or salmonella typhimurium bacterium solution.
In still another embodiment of the invention, a kind of method increased based on bacterium to the quick microscopy of bacterium is provided,
Specifically include that steps are as follows:
S1. beta-lactam antibiotic is added into sample to be tested and antibody modification magnetic ball carries out incubation processing, wherein β-
Lactam antibiotics promote tested bacteria thallus to extend, and observe convenient for subsequent microscopy under the microscope；Antibody modification magnetic ball with
Bacterium coupling after elongation；
S2. step S1. treated sample to be tested is subjected to film filtering, captured bacterium can pass through membrane filtration operation
It is trapped on filter membrane, filter membrane is then subjected to Magneto separate processing further removal impurity；
S3. by step S2., treated that filter membrane is placed in microscopically observation and counts.
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 that penicillin and its derivative, cephalosporin, monoamides ring class, carbon are green
Mould alkenes and penems enzyme inhibitor, it is preferred that the beta-lactam antibiotic includes cefalexin, aztreonam and A Mo
XiLin；Still more preferably, the beta-lactam antibiotic is aztreonam；The aztreonam addition concentration is 1~100 times
Minimal inhibitory concentration (preferably 1 times of minimal inhibitory concentration)；
Antibody modification magnetic ball preparation are as follows:
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate is cleaned with distilled water；EDC/NHS solution is added thereto, in room temperature item
It is activated under part, then carries 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, react under room temperature, Magneto separate is then molten with PBS
Liquid cleans repeatedly, and then the magnetic ball of antibody modification is added in PBS-BSA solution and is closed, then PBS solution is used in concussion processing
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 (preferably 2 μ g/mL)；
In still another embodiment of the 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 the present invention.
Embodiment
1. materials and methods
Escherichia coli O 157: H7 (ATCC 43888) and salmonella (ATCC 14028) are bought in China General Microbiological
Culture presevation administrative center.Anti- Escherichia coli antibody, anti-salmonella antibody and marked by fluorescein isothiocyanate IgG antibody are bought
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.(5 μm) of polycarbonate membrane purchases are in Shanghai Fu Xue Biotechnology Co., Ltd.Carbodiimides hydrochloric acid
Salt, the purchase of N- hydroxysuccinimide are in upper sea green source Biotechnology Co., Ltd.Cefalexin, aztreonam, Amoxicillin purchase
It buys and holds gloomy biological Co., Ltd in Jinan.Beef extract, peptone, yeast extract, agar powder purchase are 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: weighing EDC 9.6mg respectively, NHS 9.6mg is dissolved in 3mL secondary water.
0.01mol·L-1Phosphate buffer solution is prepared: weighing Na respectively2HPO4·12H2O 13.76g、NaH2PO4·
2H2O 1.79g, NaCl 9.00g, add deionized water to be settled to 1000mL.
SYBR Green Ι standard solution is prepared: SYBR Green Ι stoste being diluted 100 times with DMSO, -20 DEG C of preservations.
EDTA-2Na is prepared using liquid: weighing EDTA-2Na 18.61g, sterile purified water is added to be settled to 100mL.
LB liquid medium is prepared: tryptone 10g, yeast extract 5g, NaCl 10g are weighed respectively, it is fixed with deionized water
Hold to 1000mL, adjusts pH to 7.0.
Beef-protein medium is prepared: being weighed beef extract 3g, peptone 10g, NaCl 5g respectively, is used deionized water
It is settled to 1000mL, 20g agar is added.
3. Bacteria Culture and sample treatment
It is fallen in LB liquid medium with oese from picking Escherichia coli on inclined-plane and salmonella typhimurium single bacterium, 37
DEG C shaken cultivation 12h.After bacteria suspension is diluted 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 respectively, are added in bacteria suspension, 37 DEG C of culture 2h.Every
0.5h observes Bacteria cold shock using inverted microscope and photographs to record.The length variation 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 is cleaned once with distilled water.250 μ L EDC/NHS solution are added,
30min is activated at room temperature, and Magneto separate cleans 3 times repeatedly with PBS solution to get 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 uses 0.01molL-1PBS solution is cleaned 3 times repeatedly.The magnetic ball of antibody modification is added to 1%
It is 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
It takes 1mL bacterium solution in polystyrene sample pipe, 5min is preheated in 37 DEG C of water-baths, 10 μ L EDTA-2Na, which are added, to be made
With liquid, 10 μ L SYBR Green Ι standard solutions are rapidly added after mixing, 37 DEG C are protected from light incubation 10min, 4 DEG C of 3000r
min-1It is centrifuged 5min, 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 precipitating.
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 captured by the magnetic ball specificity of antibody modification.Captured bacterium can be trapped by membrane filtration operation
Onto filter membrane, filter membrane, which is put on Magneto separate frame, to be cleaned with PBS can further remove the impurity being trapped on filter membrane.Then will
Filter membrane is placed in microscopically observation and counts.
Results and discussion
1. the optimization of bacterium condition of growth
Beta-lactam antibiotic includes penicillin, cephalosporin and the monobactams of new development etc..Cephalo
Ammonia benzyl, aztreonam 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, aztreonam concentration
For 1 times of minimal inhibitory concentration (128 μ gmL-1) when Escherichia coli (A) and salmonella (B) thallus 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 dissolution at high concentrations.Therefore, Antibiotics and concentration optimization
For aztreonam, 1 times of minimal inhibitory concentration.
2. the characterization of magnetic ball
Magnetic ball is characterized by microscope photo.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 simultaneously with aztreonam to be incubated for.Bacterium can be special by the magnetic ball of antibody modification
Property capture.Magnetic ball is separated by Magneto separate frame, then uses 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 plate.37 DEG C of cultures are for 24 hours.Magnetic ball after closing is added to
In bacterium solution as a control group.As shown in Figure 3B, Escherichia coli (a) and salmonella typhimurium that the magnetic ball after closing is captured
(c) it is not grown on plate, the magnetic ball after illustrating closing does not have non-specific adsorption to bacterium.It is caught by the magnetic ball of antibody modification
The Escherichia coli (b) obtained and salmonella typhimurium (d) well-grown on plate, show antibody modification magnetic ball can with it is thin
Bacterium successfully combines.
3. the concentration optimization of magnetic ball surface modification antibody
The IgG antibody of FITC label is modified as model to magnetic ball surface.The concentration of fixed magnetic ball, various concentration
FITC-IgG is modified to magnetic ball surface, detects its fluorescence intensity with Fluorescence Spectrometer.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 increases.When FITC-IgG concentration is 2.0 μ g/
When mL, complex fluorescence intensity nearly reaches maximum value.And with the increase of FITC-IgG concentration, the fluorescence intensity of compound
Change it is unobvious, and as shown in Figure 3 C, when FITC-IgG antibody concentration be 2.0 μ g/mL when, fluorescence intensity is apparently higher than control group
(0 μ g/mL) shows that antibody and magnetic ball are coupled successfully, therefore selecting optimization antibody concentration is 2.0 μ g/mL.
4. the detection of Escherichia coli and salmonella in water sample
Escherichia coli and salmonella are added to the feasibility that this method is examined in water sample.It is 5,10,50 by quantity,
100 bacterium is added in water, then by LB culture medium and water sample with the volume mixture of 9:1.By aztreonam and antibody modification
Magnetic ball is added in water sample simultaneously, 37 DEG C of incubation 1.5h.By film filtering joint Magneto separate operation, filter membrane is put under microscope
Observation.Fig. 4 indicates the Escherichia coli (a) captured by magnetic ball and salmonella typhimurium (c) on glass slide.Bacterium after growth
There is bigger surface area, can be closed with more magnetic chous, to improve the capture rate of magnetic ball.Fig. 4 (b, d) is respectively by magnetic
The Escherichia coli and salmonella typhimurium of ball capture are trapped on filter membrane after film filtering and Magneto separate operation.Table 1 is big
Enterobacteria (a) and salmonella typhimurium (b) are added to the rate of recovery in water sample.As shown in 1 result of table, when the number of addition cell
When amount is 50-100, the rate of recovery is 92.00 ± 7.94%-94.67 ± 8.96%, when cell additive amount 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 detection limit of two kinds of bacteriums can satisfy the requirement of rapid detection method down to 5 cells.
Table 1
5. the method for the present invention specific assay
After Escherichia coli, salmonella typhimurium and staphylococcus aureus are dyed with SYBR Green I, while being added anti-
The magnetic ball and aztreonam 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 the magnetic ball of anti-Escherichia coli antibody modification is added, Escherichia coli it is glimmering
Luminous intensity is apparently higher than salmonella typhimurium and staphylococcus aureus.Fig. 5 b indicates that anti-salmonella typhimurium antibody is added
Afterwards, Escherichia coli can be ignored compared with salmonella typhimurium with the fluorescence intensity of staphylococcus aureus.The experiment
Show the non-specific adsorption very little of magnetic ball and this method has and compares high specific and selectivity.Not due to beta-lactam antibiotic
It can cause the growth of gram-positive bacteria, be suitable for detecting Gram-negative bacteria in this way.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (5)
1. a kind of method increased based on bacterium to the quick microscopy of bacterium, which is characterized in that comprise the following steps that
S1. beta-lactam antibiotic is added into sample to be tested and antibody modification magnetic ball carries out incubation processing, be incubated at 37 DEG C
1.5h；Wherein, beta-lactam antibiotic promotes tested bacteria thallus to extend, and observes convenient for subsequent microscopy under the microscope；It is anti-
The bacterium that body is modified after magnetic ball and elongation is coupled；The beta-lactam antibiotic is aztreonam, and the aztreonam adds concentration
For 1 times of minimal inhibitory concentration；
S2. step S1. treated sample to be tested is subjected to film filtering, captured bacterium can be cut by membrane filtration operation
It is left on filter membrane, filter membrane is then subjected to Magneto separate processing further removal impurity；
S3. by step S2., treated that filter membrane is placed in microscopically observation and counts；
The bacterium is Escherichia coli or salmonella typhimurium；
The antibody is anti-Escherichia coli antibody or anti-salmonella typhimurium antibody, and the antibody addition concentration is 0.1~5 μ
g/mL。
2. method as described in claim 1, which is characterized in that antibody modification magnetic ball the preparation method comprises the following steps:
1) carboxyl magnetic ball is taken, ultrasound, Magneto separate is cleaned with distilled water；EDC/NHS solution is added thereto, at room 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, react under room temperature, 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, then concussion processing is resuspended with PBS solution
Obtain antibody modification magnetic ball.
3. method as described in claim 1, which is characterized in that the antibody addition concentration is 2 μ g/mL.
4. application of any one of the claim 1-3 the method in quick bacteria microscopy.
5. application as claimed in claim 4, which is characterized in that the application mode is to Escherichia coli in drinking water or mouse typhus
Salmonella carries out quick microscopy.
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