CN102841198B - Method for sensitively, simply and conveniently detecting bacteria - Google Patents
Method for sensitively, simply and conveniently detecting bacteria Download PDFInfo
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- CN102841198B CN102841198B CN201210345823.9A CN201210345823A CN102841198B CN 102841198 B CN102841198 B CN 102841198B CN 201210345823 A CN201210345823 A CN 201210345823A CN 102841198 B CN102841198 B CN 102841198B
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Abstract
The invention relates to a method for detecting bacteria. The method comprises the steps as follows: coupling magnetic nanospheres and fluorescent nanospheres with mouse typhus salmonella antibodies so as to obtain mouse typhus salmonella-targeted immunologic magnetic spheres and immunologic fluorescent spheres, and adding the mouse typhus salmonella-targeted immunologic magnetic spheres and immunologic fluorescent spheres into a detection system so as to realize magnetic capture and fluorescent labeling to the mouse typhus salmonella simultaneously. About 10 CFU/mL of the mouse typhus salmonella can be detected through observation of a fluorescent microscope; and a good quantitative relation is obtained through detection of a fluorescence spectrophotometer, the linear range is 105-107 CFU/mL, and R2 is equal to 0.9994. The whole detection process is very simple, high in sensitivity and strong in specificity, and can be finished detection within 1.5 h.
Description
Technical field
The present invention relates to a kind of method of bacterial detection, belong to microbiology and chemicobiology field.
Background technology
Foodborne bacterial pathogens serious threat is to the health and lives of the mankind, there is a lot of people therefore sick, even death of being in hospital every year, and due to the infective dose of a lot of pathogenic bacteria very low (infective dose of such as enterorrhagia Bacillus coil 0157: H7 and salmonella is low to moderate 10 cells), so simply, quick, sensitive, reliably the life and health of bacterial detection to the mankind is most important.But traditional detection method, the method of such as cultivating, enzyme linked immunosorbent assay (ELISA) have the shortcomings such as length consuming time, sensitivity is low, and although the method sensitivity of PCR is high, usually needs to carry out sample pre-treatments and need professional to operate and step is more loaded down with trivial details thus limit its application.
In recent years, along with the fast development of nanosecond science and technology, increasing Application of micron is in Bacteria Detection, and show huge advantage, such as magnetic Nano material is widely used in fast Acquisition and enrich target bacterium from complex sample as a kind of separating tool, it has following advantage: magnetic Nano material has superparamagnetism, just can be handled it by one block of magnet; Specific surface area is large, can the abundant functional molecular (antibody, aptamers, Avidin, agglutinin etc.) of coupling; And relative to the interfacial reaction of ELISA, there is reaction kinetic characteristics faster; Be easy to realize and various detection means be used in conjunction, as the method with traditional cultivation, PCR, electrochemical method, optical detecting method etc. are used in conjunction, and greatly reduce detection time and detectability.But in these detection methods, major part all will catch and mark and separately carry out, and step is more, and testing process is also comparatively loaded down with trivial details.
Summary of the invention
Technical matters to be solved by this invention is the method providing the easy bacterial detection of a kind of rapid sensitive.
The present invention is by the antibody of target bacteria on magnetic nano-balls and fluorescent nanosphere coupling, thus the biomolecular obtained target bacteria target and immunofluorescence ball, then they are put in system to be checked simultaneously, thus achieve simultaneously the magnetic of target bacteria is caught and fluorescence labeling, by detecting the fluorescence signal bacterial detection of magnetic ball-bacterium-fluorescent balls compound.
Concrete technical scheme is:
1, the antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) (BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
2, biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, Magneto separate also washs, and finally adds physiological saline and evenly resuspended, obtains magnetic ball-bacterium-fluorescent balls compound;
3, fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
Wherein in step 1, the antibody of target bacteria is connected to magnetic nano-balls and fluorescent nanosphere surface, is specially: by EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride)/NHS (N-hydroxy-succinamide) activation method by NH
2-PEG-COOH is coupled to magnetic nano-balls and fluorescent nanosphere is surperficial as spacer (bridge), then is connected with the antibody of target bacteria by EDC/NHS activation method.。
Described magnetic nano-balls, surface, with-COOH, is by oil-soluble nano-γ-Fe
2o
3be embedded in styrene-propene acid amides compolymer/nano ball and prepare.
Described fluorescent nanosphere, surface, with-COOH, is oil-soluble CdSe/ZnS quantum dot (QDs) be embedded in styrene-propene acid amides compolymer/nano ball to prepare.
Described target bacteria refers generally to food-borne pathogens, as salmonella, Escherichia coli, shigella dysenteriae, staphylococcus aureus etc.
Based on said method, the present invention utilizes coupling to have
s.
typhimuriumthe biomolecular of monoclonal antibody catch
s.
typhimurium, and utilize coupling to have
s.
typhimuriumthe immunofluorescence ball identification of monoclonal antibody
s.
typhimurium, realize catch with mark while carry out, by fluorescent microscope or fluorescence spectrophotometer, magnetic ball-bacterium-fluorescent balls compound is detected.
The inventive method utilizes biomolecular and immunofluorescence ball to realize catching while target bacteria and marking, bacterial detection is carried out by the fluorescence signal detecting magnetic ball-bacterium-fluorescent balls compound, step is very simple, without the need to carrying out pre-service or preenrichment to sample, and highly sensitive, specificity is good.By fluorescence microscope, the salmonella typhimurium of about 10 CFU/mL can be detected; Detected by fluorescence spectrophotometer, obtain good quantitative relationship, the range of linearity 10
5~ 10
7cFU/mL, R
2=0.9994.Whole testing process is very simple, and highly sensitive, high specificity, can complete detection in 1.5 h.
Accompanying drawing explanation
Fig. 1 is transmission electron microscope picture (A) and magnetic hysteresis loop sign (B) of magnetic nano-balls.
Fig. 2 is the transmission electron microscope picture (A) of fluorescent nanosphere, shows fluorescent microscopy images (B) and fluorescence spectrum figure (C, dotted line is the fluorescence spectrum figure of quantum dot, solid line is the spectrogram of fluorescent balls) and the graph of a relation (D, inner illustration is the linear fit of fluorescent balls low concentration situation) of its fluorescence intensity and its concentration.
Fig. 3 detects the schematic diagram of salmonella typhimurium.
Fig. 4 detects the fluorescent microscope picture of salmonella typhimurium: be respectively 0 from the concentration of A ~ E bacterium, 10,50,100,1000,10000CFU/ml, F to catch by biomolecular one and the empty enlarged drawing of typical bacterium of immunofluorescence ball mark.
Fig. 5 is the fluorescence spectrum figure (A) and and the linear fit (B) of fluorescence intensity and bacterial concentration that detect salmonella typhimurium.
Fig. 6 is detection 10
7cFU/mL salmonella typhimurium (
s.
typhimurium) and physiological saline and blank, Escherichia coli (
e. coli), Shigella flexneri (
s. flexneri) and with bare ball MNS and FNS of non-coupled antibody and the fluorescence intensity histogram of salmonella typhimurium effect.
Fig. 7, for detecting salmonella typhimurium (A), physiological saline (B), Escherichia coli (C), Shigella flexneri (D) and the bare ball MNS/FNS of non-coupled antibody and the laser confocal microscope picture of salmonella typhimurium effect (E), is followed successively by light field; Hoechst 3342: excite: 405 nm, launch: 447 ± 30 nm; FNS: excite: 488 nm, launch: 565 ± 10 nm; Superposition: the superposition of two glimmering field passages.Each group of bacterium is all dyeed with Hoechst 33342 before detection.
Fig. 8 is the result that analog sample detects: the top of figure is divided into the shows fluorescent microscopy images detected in physiological saline, milk, hyclone, urine; Figure A is corresponding fluorescence spectrum figure, schemes the linear relationship chart that B is fluorescence intensity and the bacterial concentration detected in milk.
Embodiment
Following examples only for further illustrating the present invention, but should not be construed as limitation of the present invention.
embodiment 1 salmonella typhimurium
s.
typhimuriumdetection
Below with salmonella typhimurium
s.
typhimuriumfor example, the inventive method is described in detail.
one, method
1. the preparation of biomolecular and immunofluorescence ball
The preparation of magnetic nano-balls (MNS) and fluorescent nanosphere (FNS) carries out on the basis that fluorescence magnetic dual-functional nanometer ball is prepared in this laboratory.Namely by ultrasonic swelling method, hydrophobic interaction is utilized, by oil-soluble magnetic nanoparticle γ-Fe
2o
3or the hydrophobic cavity that quantum dot (CdSe/ZnS QDs) is embedded into styrene-propene acid amides compolymer/nano ball (Pst-AAm-COOH) of surface carboxyl groups is inner, thus obtains MNS and FNS.The diameter of nanosphere is about 250nm.Before nanosphere surface connects antibody, covalent coupling is first utilized to introduce NH
2-PEG-COOH (MW 3400) is as bridge (spacer): get about 5 mg MNS or FNS, be distributed in 0.01 M pH 6.8 PBS, and add EDC/NHS and make its ultimate density be 100 mM, activate 0.5 h, (MNS passes through magnetic separation to wash three times with 0.01 M pH 7.2 PBS, FNS passes through centrifuge washing), be then distributed in 1 mL 0.01 M pH 7.2 PBS, add about 2 mg NH
2-PEG-COOH (MW 3400) reaction four hours, then washs five times with 0.01 M pH 7.2 PBS and removes unreacted PEG.The introducing of antibody is also undertaken by EDC/NHS activation method, and step is identical with coupling PEG, just the PEG of 2 mg has been changed into the antibody of the salmonella typhimurium of about 50 μ g.Namely obtain the biomolecular (IMNS) to salmonella typhimurium target and immunofluorescence ball (IFNS), with 1% BSA close be placed in 4 ° of C refrigerators stand-by.
2. salmonella typhimurium
s.
typhimuriumdetection
By 0.32 mg IMNS, 2.61 mg IFNS join in bacterial suspension simultaneously and (by the method for gradient dilution, obtain each dilution bacterial suspension.), be placed on shaking table, 37 ° of C, 120 rpm hatch 60min, and detection system 0.1% Skim Milk (skimmed milk power)-0.05% Tween 20 (Tween-20) closes.Then immune complex passes through Magneto separate, four times are washed with 0.1% Skim Milk-0.05% Tween 20-0.9% NaCl, 0.9%NaCl washing once, finally be distributed to 300 μ L 0.9% NaCl fluorescence spectrophotometer to detect, or be distributed to 20 μ L0.9% NaCl and be enriched to the little bottom land of PDMS (diameter ~ 3 mm) and detect with fluorescent microscope.
3. specificity experiments
Adopt physiological saline, Escherichia coli (
e. coli), Shigella flexneri (
s. flexneri) as a control group, in addition, MNS and FNS not having a coupled antibody also for
s.
typhimuriumeffect in contrast.Test according to step 2 respectively.Detect for fluorescent microscope, bacterium carries out dyeing with Hoechst 33342 in advance, and (bacterial concentration is 10
7cFU/mL, Hoechst 33342 concentration be 30 μ g/mL, 37 ° of C, 30 min), wash with PBS and be distributed to 0.1% Skim Milk-0.05% Tween 20-0.9% NaCl for 3 times, then IMNS and IFNS is added detection system and catch and mark bacterium.Immune complex also, after washing, drips on slide and uses confocal microscopy by Magneto separate.
4. repeated experiment
Be 1 × 10 by concentration
6the bacterial suspension of CFU/mL, with a collection of by step 1 prepare IMNS and IFNS detect, repeat experiment 5 times according to step 2, record the fluorescent value of each testing result, calculate batch in relative standard deviation Intra-assay CV.Meanwhile, IMNS and IFNS prepared with 5 kinds of different batches detects above-mentioned sample, record testing result fluorescent value, calculate batch between relative standard deviation Inter-assay CV.
5. manual simulation's pattern detection
Bacterial suspension is joined in the complex dielectricss such as milk, hyclone, urine, according to step 2, it is detected, and with not adding the sample of bacterium in contrast, and desirable detection system (physiological saline) compares testing result.
two, result
1. the sign of magnetic nano-balls and fluorescent nanosphere
Styrene-propene acid amides compolymer/nano ball is synthesized by agalactosis polymerization in aqueous phase, has hydrophilic surface and hydrophobic cavity.Oil-soluble γ-Fe
2o
3or QDs can enter in the hydrophobic cavity of nanosphere by hydrophobic interaction in normal butyl alcohol, chloroform (volume ratio: 95:5) mixed solvent, and the transmission electron microscope picture of MNS (Fig. 1 A) and FNS (Fig. 2 A) shows γ-Fe
2o
3or QDs is well dispersed in nanosphere.By the magnetic hysteresis loop (Fig. 1 B) of MNS can find out the coercive force of MNS and remanent magnetism almost nil, show that MNS at room temperature presents good superparamagnetism.The shows fluorescent microscopy images (Fig. 2 B) of FNS and fluorescence spectrum figure (Fig. 2 C) show that FNS is very well dispersed and have excellent photoluminescent property.And the graph of a relation of the fluorescence intensity of FNS and its concentration (Fig. 2 D) shows fluorescence intensity and its concentration (0.004 mg/mL ~ 0.875 mg/mL) linear correlation (R of FNS
2=0.996), this also illustrates that the FNS of investment synthesis has good homogeneity from another point of view, and provides possibility for quantitatively detecting.
2. salmonella typhimurium
s.
typhimuriumdetection
Detect salmonella typhimurium principle and see Fig. 3, IMNS and IFNS drops into detection system simultaneously, achieves simultaneously and catches and fluorescence labeling the magnetic of salmonella typhimurium, and then product fluorescence spectrophotometer and fluorescent microscope detect.In order to realize larger concentration effect, immune complex is dispersed in the physiological saline of 20 μ L by we, then the PDMS sulculus (be bonded to by PDMS on cover glass and obtain) instilling diameter about 3 mm is inner, magnet is put into little trench bottom, thus immune compound is attracted to bottom land, then observe with fluorescence inverted microscope, the sulculus of diameter 3 mm about has 200 visuals field under 100 × object lens, namely we find object in these 200 visuals field---be marked with the bacterium of fluorescent balls, we find that this method can detect the salmonella typhimurium of about 10 CFU/mL, see Fig. 4.When immune complex fluorescence spectrophotometer detects (Fig. 5), find that positive presents obvious FNS emission peak, and negative sample does not have obvious FNS fluorescence signal, and its fluorescence intensity and bacterial concentration are 10
5~ 10
7cFU/mL presents good quantitative relationship (R
2=0.9994).
3. specificity
The reliability of a method needs to evaluate by its specificity and selectivity, and therefore a series of control experiment is very important.IMNS and IFNS respectively with physiological saline, Escherichia coli (
e. coli), Shigella flexneri (
s. flexneri) interact, specificity and the selectivity of checking this method are tested in MNS and FNS and salmonella typhimurium effect in contrast.Fig. 6 detects 10 with fluorescent spectrometry
7cFU/mL salmonella typhimurium and physiological saline and blank, Escherichia coli (
e. coli), Shigella flexneri (
s. flexneri), and with the fluorescence intensity histogram that MNS and FNS and salmonella typhimurium effect obtain, find yin and yang attribute contrast obviously.Fig. 7 is the result with confocal laser scanning microscope.Each group of bacterium is all dyeed with Hoechst 33342, then detects with IMNS/IFNS, finds to only have positive group to capture bacterium, and be combined with fluorescent balls around bacterium, and have agglutination phenomenon to a certain degree, and the magnetic ball of negative group is very well dispersed, does not all find the bacterium be captured.Prove that IMNS/IFNS specificity can catch and detect salmonella typhimurium by spectrogram and microscope photograph.
4. repeatability
In order to evaluate the reappearance of the method, determine the method batch in relative standard deviation (Intra-assay CV) and batch between relative standard deviation (Inter-assay CV), calculating Intra-assay CV uses IMNS and IFNS of same batch to detect, and calculates its relative standard deviation after repeating experiment 5 times.Calculate Inter-assay CV, with IMNS and IFNS of five kinds of different batches, detection record fluorescence intensity is carried out to sample, calculate its relative standard deviation, calculate Intra-assay CV and Inter-assay CV is respectively 5.2% and 7.2%, show that this method reappearance is better, method is reliable.
5. manual simulation's pattern detection
In reality detects, the usual more complicated of sample environment, in order to investigate the application of this detection method in complex system, we design artificial analog sample, bacterial suspension is mixed with complex samples such as milk, hyclone, urines, then detect by this method, testing result, as Fig. 8, illustrates that this method is expected to be applied to complicated actual sample.
By above every detection, prove that the inventive method can be applied to the detection of salmonella typhimurium, simple to operately fast, only need a step namely complete catching and marking target bacteria; Highly sensitive, detectability can reach about 10 CFU/mL; Reproducible and there is good selectivity and specificity, common Escherichia coli, Shigella flexneri not Interference Detection.
Claims (5)
1. a method for bacterial detection, is characterized in that, comprises the steps:
1) antibody of target bacteria to be connected to magnetic nano-balls and fluorescent nanosphere surface, and to close with the magnetic nano-balls of bovine serum albumin(BSA) antibody to coupling and fluorescent nanosphere, obtain biomolecular and immunofluorescence ball;
2) biomolecular and immunofluorescence ball are joined in solution to be measured, hatch and biomolecular, immunofluorescence ball and target bacteria are fully combined, then Magneto separate washing, finally add physiological saline and evenly resuspended, obtain magnetic ball-bacterium-fluorescent balls compound;
3) fluorescent microscope or fluorescence spectrophotometer is utilized to detect magnetic ball-bacterium-fluorescent balls compound.
2. method according to claim 1, is characterized in that, wherein in step 1, the antibody of target bacteria is connected to magnetic nano-balls and fluorescent nanosphere surface, is specially: by EDC/NHS activation method first by NH
2-PEG-COOH is coupled to nanosphere surface as bridge, then is connected with the antibody of target bacteria by EDC/NHS activation method.
3. method according to claim 1 and 2, is characterized in that, described magnetic nano-balls surface, with-COOH, is by oil-soluble nano-γ-Fe
2o
3be embedded in styrene-propene acid amides compolymer/nano ball and prepare.
4. method according to claim 1 and 2, is characterized in that, described fluorescent nanosphere, and surface, with-COOH, is be embedded in styrene-propene acid amides compolymer/nano ball by oil-soluble CdSe/ZnS quantum dot to prepare.
5. method according to claim 1 and 2, is characterized in that, described target bacteria is salmonella typhimurium.
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| CN103543260A (en) * | 2013-07-30 | 2014-01-29 | 中国检验检疫科学研究院 | Method for detecting biomacromolecule based on magnetic separation-quantum dot immunofluorescence sensing and reagent preparation method |
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| CN110699241A (en) * | 2019-10-28 | 2020-01-17 | 军事科学院系统工程研究院卫勤保障技术研究所 | Automatic disinfection effect rapid evaluation device and method |
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| CN1289628C (en) * | 2003-10-29 | 2006-12-13 | 武汉大学 | Multifunctional fluorescent, magnetic nano material and preparation method |
| US20080135490A1 (en) * | 2005-01-07 | 2008-06-12 | Board Of Trustees Of The University Of Arkansas | Quantum dot biolabeling and immunomagnetic separation for detection of contaminants |
| CN1869692A (en) * | 2005-05-23 | 2006-11-29 | 武汉大学 | Three-function nano-ball |
| CN101721964A (en) * | 2009-11-12 | 2010-06-09 | 同济大学 | Method for preparing shell-core micrometer/nanometer spheres capable of preventing functional materials |
| CN101865919A (en) * | 2010-04-29 | 2010-10-20 | 上海师范大学 | Method for rapidly detecting and screening Enterobacter sakazakii |
| CN102085380B (en) * | 2010-12-30 | 2012-09-05 | 成都西南交大科技园管理有限责任公司 | Preparation method of nano magnetic particles for detection and treatment of coronary heart diseases |
| CN102590506A (en) * | 2012-02-16 | 2012-07-18 | 上海师范大学 | Method for rapidly detecting and screening staphylococcus aureus |
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