CN107345961B - Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii - Google Patents
Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii Download PDFInfo
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Abstract
The present invention relates to a kind of methods based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii, according to the virulence gene ompA of Enterobacter sakazakii, it designs loop-mediated isothermal amplification (LAMP) primer (SEQ ID NO:1-4), in conjunction with nano enzyme nucleic acid test strip, a kind of Enterobacter sakazakii detection method based on LAMP nanometers of enzyme sensors is established.This method can be successfully used to distinguish living bacterial cells and dead bacterium cell, to the Monitoring lower-cut of Enterobacter sakazakii up to 10CFU/mL.
Description
Technical field
The present invention relates to bio-sensing detection technique fields, specifically, being related to a kind of based on nucleic acid chromatography bio-sensing
The method of technology detection Enterobacter sakazakii.
Background technique
Enterobacter sakazakii (Cronobacter sakazakii category) is a kind of Gram-negative, by peritrichous movement, nonspore-bearing simultaneous
Property anaerobic bacteria.It is a kind of conditioned pathogen of Enterobacter, which can lead to baby (especially
Newborn) meningitis, enterocolitis and bacteremia, the death rates of reported cases reaches 20%-50%, and survivor often leaves sternly
The nervous system disease of weight.In recent years to food (milk powder, chocolate, cereal preparation, potato, pasta etc.) processing factory original
Enterobacter sakazakii pollution rate the study found that Enterobacter sakazakii is widely distributed in nature in material, production environment and family.?
Breaking out in event for a lot of Enterobacter sakazakiis finds that the infection of newborn Enterobacter sakazakii is closely related with infant formula.
Traditional method of detecting bacterium is mainly according to physiological and biochemical property, but traditional detection method is needed by preceding increasing
Bacterium, the separation of selective plate, Biochemical Identification, need 5-7 days from definitive result is sampled to, detection cycle is long, grasps
Make cumbersome, heavy workload；Using the specificity of antigen-antibody reaction, bacterium is identified, has the history of over half a century,
But the screening of microbial antibodies is very complicated, and final detection specificity is not high；Molecular Biological Detection technology is not
It is disconnected to improve and develop, the problems such as traditional detection method experimental implementation is cumbersome, time-consuming is overcome, but also carrying out for microorganism
Rapid detection method rapidly developed, but being to be not easy the shortcomings that molecular biology method analyzes result.With existing
For the development of biotechnology, more faster than conventional method, more sensitive biosensor new technology is in field of detection of food safety
Show up prominently.Biosensor has the advantages that high specificity, high sensitivity, can simplify analysis detection step, shortens analysis
Time, it is often more important that make it possible on-line real-time measuremen, easy to carry and field work is obtained in field of food safety
Faster development.In recent years, the development and application of numerous new materials, new technology just has become a hot topic of research, and is biosensor
Development be filled with new vitality, show huge application potential in microorganism detection field.Therefore Enterobacter sakazakii is established
Reliably, quick sensor detecting method, the method that can especially identify life or death bacterium, in daily monitoring, market screening etc.
Aspect is of great significance.
Summary of the invention
The object of the present invention is to provide a kind of methods based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii.
Present inventive concept is as follows: developing a kind of quick and super sensitivity detection method of Enterobacter sakazakii, can especially reflect
The method for fixing viable bacteria is established and is based on nitrine propidium iodide (PMA), loop-mediated isothermal amplification (LAMP) and nanometer enzyme test peper
Living stems continuous cascade nanometer enzyme biologic sensor.In LAMP reaction, using fluorescein (FITC) modification and raw
The ompA gene of the primer measurement Enterobacter sakazakii of object element (BIO) modification.It combines PMA with LAMP, is applied to the rugged intestines of life or death slope
The separation of bacillus.Then, using nanometer enzyme probe immunochromatography item (nano enzyme item) of the preparation based on magnetic-particle for detecting
Amplified signal detects by visual observation or carries out result interpretation using bar graph-type readout or quantify, is on-site test Enterobacter sakazakii
Viable bacteria provides quick, hypersensitive and convenient and fast tool.
In order to achieve the object of the present invention, present invention firstly provides for detecting Enterobacter sakazakii (Enterobacter
Sakazakii LAMP primer group), including (SEQ ID NO:1-4):
Outside forward primer F3:5 '-TGGTCCCAGTTCCACGATA-3 '；
Outside reverse primer B3:5 '-ACGCCCTGAGCTTTGAAAG-3 '；
Inside forward primer FIP:5 '-TAACCTGGTAACCACCGAACGCTATTCCTAACGACGGTCCGA-3 '；
Inside reverse primer BIP:5 '-GTACGTTGGCTTCGAAATGGGCCAGTGTCGCCTTTATACGGC-3 '.
Wherein, 5 ' the end label biotins of primers F IP, 5 ' the end mark fluorescent element FITC of primer BIP.
The present invention also provides a kind of nanometer of enzymatic nucleic acid chromatograph test strip, the preparation method of the test strips includes following step
It is rapid:
1)Fe3O4The preparation of magnetic particle；
2) preparation of nanometer enzyme probe (MNP): by Fe3O4Magnetic particle is incubated for biotin secondary antibody, obtains biotin two
Anti- nanometer enzyme probe；
3) assembling of nanometer enzymatic nucleic acid chromatograph test strip: the test strips include sample pad, bonding pad, nitrocellulose filter
And absorption pad, wherein the nitrocellulose filter is equipped at least 1 detection line and 1 nature controlling line；It is fixed on the bonding pad
There is biotin secondary antibody nanometer enzyme probe；
1. marking detection line and nature controlling line on nitrocellulose filter with FITC antibody and biotin antibody respectively, dry；
2. successively by above-mentioned sample pad, bonding pad, the nitrocellulose filter with detection line and nature controlling line and absorption pad
It is pasted on bottom plate (plastics lining board), completes the assembling of test strips.The structural schematic diagram of assembled test strips is shown in Fig. 5.
Fe is synthesized using hydro-thermal method in step 1)3O4Magnetic particle, specifically: by 0.6-0.8g FeCl3·6H2O is dissolved in
In 20mL ethylene glycol, 1.5-2.0g sodium acetate is then added, is vigorously stirred 30-40min, is then sealed in autoclave, 200 DEG C
Heat 16-18h；Magnetic granular product with ethanol washing several times, and it is dry at 60 DEG C；Dichloroethanes and N- hydroxysuccinimidyl acyl is sub-
Each 5-8mg of amine is dissolved in 1mL deionized water by being vortexed, and mixed liquor is made, and mixed liquor then is added in 5-8mg magnetic particle
In, it is incubated for 30-40min at room temperature, then magnetic particle is collected with magnet, with milli-Q water twice to get Fe3O4Magnetic particle.
Step 2) specifically: the biological antibody of 100 μ g/mL of concentration is added in the sodium-acetate buffer of 50mM pH 6.0,
Then with the Fe of 5-8mg3O4Magnetic particle mixing, by mixture vortex mixed, 4 DEG C of overnight incubations；Two are washed with the PBS liquid of pH7.0
Then secondary mixture is incubated at room temperature 30min in the Tris buffer of 50mM pH7.2；It is washed again with the PBS liquid of pH7.0, i.e.,
Obtain biotin secondary antibody nanometer enzyme probe.
Detection line is located on the position away from nitrocellulose filter lower edge 1.1cm in step 3), and nature controlling line is located at away from nitric acid
On the position of cellulose membrane lower edge 1.6cm.The distance between detection line and nature controlling line are 4.5mm.By 1.0 μ L/cm by FITC
Antibody and biotin antibody are sprayed on respectively in the detection line and nature controlling line of nitrocellulose filter.Wherein in detection line and nature controlling line
The concentration of coated antibody is 0.5-2mg/mL.The optium concentration of antibody is 1mg/mL.
Biotin antibody used in the present invention is purchased from Sigma company, and goods number B7653, FITC antibody is purchased from Sigma
Company, goods number F5636.Biotin secondary antibody is sheep anti-mouse igg.Nitrocellulose filter used is Millipore135S.
In a preferred embodiment of the present invention, the nanometer enzymatic nucleic acid chromatograph test strip prepare it is as follows:
Fe is synthesized using hydro-thermal method in step 1)3O4Magnetic particle, specifically: by 0.6g FeCl3·6H2O is dissolved in 20mL
In ethylene glycol, 1.5g sodium acetate is then added, is vigorously stirred 30min, is then sealed in autoclave, 200 DEG C of heating 16h；Magnetic
Granular product with ethanol washing several times, and it is dry at 60 DEG C；Dichloroethanes and each 5mg of n-hydroxysuccinimide are passed through into whirlpool
Rotation is dissolved in 1mL deionized water, and mixed liquor is made, and then 5mg magnetic particle is added in mixed liquor, is incubated for 30min at room temperature,
Then magnetic particle is collected with magnet, with milli-Q water twice to get Fe3O4Magnetic particle.
Step 2) specifically: the biotin secondary antibody of 100 μ g/mL of concentration is added to the sodium-acetate buffer of 50mM pH6.0
In, then with the Fe of 5mg3O4Magnetic particle mixing, by mixture vortex mixed, 4 DEG C of overnight incubations；It is washed with the PBS liquid of pH7.0
Then mixture twice is incubated at room temperature 30min in the Tris buffer of 50mM pH7.2；It is washed again with the PBS liquid of pH7.0,
Obtain biotin secondary antibody nanometer enzyme probe.Finally nanometer enzyme probe is distributed in the 5%BSA-PBS solution of 1mL.It uses
JEOL 2000FX 200kV transmission electron microscope (TEM) observes the partial size of nanometer enzyme probe, and the size distribution display of MNP is flat
Equal diameter is 200nm, can be used for the building of nanometer enzyme sensor.
By sample pad, the bonding pad for being fixed with biotin secondary antibody nanometer enzyme probe, the nitric acid with detection line and nature controlling line
Cellulose membrane and absorption pad are successively pasted on bottom plate, that is, complete the assembling of test strips.
The present invention also provides the methods based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii, including following step
It is rapid:
S1, sample to be tested DNA is extracted, using DNA as template, carries out LAMP-PCR using above-mentioned LAMP primer group and expand instead
It answers；
S2, the LAMP-PCR amplified production for taking 10 μ L step S1, are added drop-wise to above-mentioned receive after mixing with 50 μ L reaction buffers
In the sample pad of rice enzymatic nucleic acid chromatograph test strip, after 5-20min (preferred reaction time is not less than 15min), in p-wire and matter
It controls and 2 drop substrate buffer solutions is added dropwise on line, then observe by the naked eye colour developing situation after reacting 5min, interpretation result: negative reaction:
Nature controlling line colour developing, detection line do not develop the color；Positive reaction: nature controlling line, detection line develop the color；Failure reaction: if nature controlling line does not develop the color,
Then detect failure or test strips failure.
For quantitative measurment, using the optics of the portable strip reader record strip band combined with band reader software
Intensity calculates the content of Enterobacter sakazakii in sample to be tested according to optical strength.
Wherein, reaction buffer described in step S2 are as follows: 4 × SSC, 0.2m/m% Tween-20.
Substrate buffer solution used in the present invention is that commercial reagents (are purchased from the limited public affairs of Beijing Zhong Shan Golden Bridge biotechnology
Department), contain 20 × DAB and 20 × H2O2。
In the present invention, the system of LAMP-PCR amplified reaction are as follows: 1 × Bst Thermal Buffer, 0.6M Betaine,
0.5mM dNTPs solution, 1.6 μM of FIP, 1.6 μM of BIP, 0.2 μM of F3,0.2 μM of B3,3.6mM MgSO4, 8U Bst DNA
Polymerase Large fragment, 2 μ L templates, ddH2O complements to 25 μ L.
LAMP-PCR response procedures are as follows: 65 DEG C of 30min, then 85 DEG C of 3min.
Before step S1, further include the steps that carrying out sample to be tested the processing of nitrine propidium iodide, specifically: to 1 μ L
The nitrine propidium iodide of final concentration of 10 μ g/mL is added in sample to be tested containing viable bacteria or hot inactivation of bacterial, is protected from light incubation
Then sample cell is remotely from light source 20cm using 500W halogen light source and level is put by 5min by sample exposure 5min
It sets on ice, shakes sample cell every 30s to keep the uniform exposure to light source；Then DNA extraction is carried out.
The present invention further provides contain the LAMP primer group for detecting Enterobacter sakazakii and/or the nano enzyme
The kit of nucleic acid chromatograph test strip.
The invention has the following advantages that
(1) present invention develops the nanometer enzyme biologic sensor that PMA, LAMP are combined with nanometer enzyme test peper for the first time.
(2) nanometer enzyme test peper is successfully used to distinguish living bacterial cells and dead bacterium cell for the first time.
(3) nanometer enzyme biologic sensor of the invention can be used for the field diagnostic test of Enterobacter sakazakii viable bacteria.
(4) one group of new LAMP primer is provided, can be used for detecting the ompA gene of Enterobacter sakazakii.
(5) nanometer enzyme sensor is to the Monitoring lower-cut of Enterobacter sakazakii up to 10CFU/mL.
Detailed description of the invention
Fig. 1 is Enterobacter sakazakii LAMP amplification in the embodiment of the present invention 1；Wherein, M is DNA Marker, swimming lane 1-3
For Enterobacter sakazakii positive amplification, swimming lane 4 is negative amplification.
Fig. 2 is the optimization in the embodiment of the present invention 2 to biosensor；Wherein, (A) membrane material is to biosensor peak face
Long-pending influence；(B) influence of the FITC antibody concentration to biosensor peak area in detection line；(C) dosage of nanometer enzyme probe
Influence of the volume to the peak area of biosensor；(D) influence of the reaction time to the peak area of biosensor.
Fig. 3 is that performance of biosensor analyzes result figure in the embodiment of the present invention 3；Wherein, (A) sensor reproducibility is analyzed
Result figure.(B) sensor specificity analyzes result figure.Sample is respectively from left to right: distilled water, other enterobacteria DNA cloning
Product, other non-Enterobacter sakazakii bacterial strain DNA cloning products, dead Enterobacter sakazakii bacterial strain DNA cloning product, Enterobacter sakazakii living
Bacterial strain DNA cloning product；(C) sensor stability analyzes result figure.It is from left to right the survey with a collection of sensor every other week
Test result.
Fig. 4 is the linearity curve of Enterobacter sakazakii detection in the embodiment of the present invention 4: (Enterobacter sakazakii is dense with lg for peak area
Degree) variation.
Fig. 5 is the structural schematic diagram of nanometer enzymatic nucleic acid chromatograph test strip of the present invention.Wherein, 1- sample pad, 2- cellulose nitrate
Plain film, 3- bonding pad, 4- absorption pad, 5- p-wire, 6- nature controlling line, 7- bottom plate.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
Method of the embodiment 1 based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii
1, experimental material
Enterobacter sakazakii used and non-Enterobacter sakazakii bacterial strain information are shown in Table 1 in the present embodiment.
The information of Enterobacter sakazakii used in table 1 and non-Enterobacter sakazakii
The specificity of nanometer enzyme sensor is measured using Enterobacter sakazakii and other bacterium bacterial strains.To own before use
Bacterial strain is stored in -80 DEG C 20% (v/v) glycerite.It is then incubated overnight activation in LB culture medium.By dividing
Light photometry measures Enterobacter sakazakii concentration.
2, Enterobacter sakazakii genome extracts
Using the bacterial genomes DNA extraction kit of New Industry company, the specific steps are as follows:
(1) inoculum 1.5mL, 12000g is taken to be centrifuged 1min, as far as possible exhaustion supernatant.
(2) 200 μ L solution As are added into bacterial precipitation, oscillation sufficiently suspends to thallus.
(3) Proteinase K of 20 μ L10mg/mL is added into pipe, mixes well, during which can overturn centrifuge tube and mix number,
Until treatments of the sample is complete.Digesting complete mark is that liquid is limpid and sticky.
(4) 2000 μ L solution Bs are added into pipe, mix well.Such as there is white precipitate, 75 DEG C of 15-30min can be placed in,
Precipitating can disappear, and not influence subsequent experimental.
(5) 220 μ L dehydrated alcohols are added into pipe, mix well, at this time it is possible that flocculent deposit, does not influence DNA
Extraction, solution and flocculent deposit can be all added in adsorption column, stand 2min.
(6) 12000g is centrifuged 1min, and adsorption column is put into collecting pipe by waste liquid.
(7) 700 μ L rinsing liquids are added into adsorption column, 12000g is centrifuged 1min, and adsorption column is put into collecting pipe by waste liquid
In.
(8) 500 μ L rinsing liquids are added into adsorption column, 12000g is centrifuged 1min, and adsorption column is put into collecting pipe by waste liquid
In.
(9) 12000g is centrifuged 2min, and adsorption column is placed in room temperature or 50 DEG C of incubators are placed several minutes, is removed residual in adsorption column
The rinsing liquid stayed.
(10) adsorption column is put into a clean centrifuge tube, to the hanging 500-200 μ L that is added dropwise in adsorbed film center through 75
The eluent of DEG C water-bath preheating, is placed at room temperature for 2min, and 12000g is centrifuged 2min.
(11) centrifugation gained eluent adds in adsorption column, is placed at room temperature for 2min, and 12000g is centrifuged 2min, can be obtained
The bacterial genomes DNA of high quality.
3, nitrine propidium iodide is handled
By 1 μ L viable bacteria or hot inactivation of bacterial, the PMA of final concentration of 10 μ g/mL is added.Sample is protected from light and is incubated for 5min, and
Mixing makes PMA penetrate into dead cell.Then use 500W halogen light source by sample exposure 5min.Sample cell is remotely from
It is at light source 20cm and horizontal positioned on ice to avoid excessive heating.Every 30s shakes sample cell to keep to the uniform sudden and violent of light source
Dew.Using the genomic DNA of the bacterial genomes DNA extraction kit separation Enterobacter sakazakii of New Ind μ stry company, mention
The genomic DNA taken carries out LAMP reaction immediately after preparation.In the heat inactivation Enterobacter sakazakii sample of PMA processing, do not have
Genomic DNA is extracted, and sample cannot use LAMP to be expanded.And not using the life or death bacterium genomic DNA of PMA processing
It can be carried out expanding.As it can be seen that handling the amplified band for eliminating dead bacterium by PMA, the amplified band of viable bacteria is only left.Thus
Prove that PMA processing can effectively eliminate the DNA signal of dead bacterium.
4, design of primers
According to the conserved region for the Enterobacter sakazakii gene ompA that document is announced, drawn by the mediation of Japanese Rong Yan Co., Ltd. ring
Object Photographing On-line software LAMP primer designing software primerexplorer V 4.0 (https: //
Primerexplorer.jp/lamp4.0.0/index.html gene design primer, including 2 outer primers F3, B3 and 2) are directed to
Inner primer FIP, BIP (table 2).In 5 ' the end label biotins of FIP, 5 ' the end mark fluorescents of BIP are plain (FITC).Primer is by upper
The synthesis of Hai Yingweijieji biology Co., Ltd, is used for subsequent screening and optimizing.
Primer sequence used in table 2LAMP
5, the amplification of Enterobacter sakazakii genome
(table 3) is expanded to Enterobacter sakazakii genome using ring mediated isothermal amplification method.
Table 3LAMP reaction system
LAMP response procedures are as follows: in 65 DEG C of reaction 30min, inactivate enzyme in 85 DEG C of reaction 3min.This experiment uses every pipe
The system of 25 μ L, wherein Enterobacter sakazakii genome is not added as negative control group.5 μ L of amplified production is taken, with 1 μ L loading buffer
Liquid is uniformly mixed, and carries out electrophoresis with 2% Ago-Gel, typical DNA cloning band (Fig. 1) occurs in Enterobacter sakazakii, and non-slope is rugged
Enterobacteria bacterial strain does not generate any band, thus proves that the primer pair Enterobacter sakazakii of design has 100% specificity.
6, the preparation of magnetic particle and nanometer enzyme probe
Magnetic particle is synthesized according to hydro-thermal method.Specifically, by 0.6g FeCl3·6H2O is dissolved in 20mL ethylene glycol, then
1.5g sodium acetate is added, is vigorously stirred 30min, is then sealed in autoclave, 200 DEG C of heating 16h；Magnetic granular product ethyl alcohol
It washs several times, and dry at 60 DEG C；Dichloroethanes and each 5mg of n-hydroxysuccinimide are dissolved in 1mL by being vortexed
In ionized water, mixed liquor is made, then 5mg magnetic particle is added in mixed liquor, is incubated for 30min at room temperature, is then received with magnet
Collect magnetic particle, with milli-Q water twice to get Fe3O4Magnetic particle.
The biotin secondary antibody (sheep anti-mouse igg) of 100 μ g/mL of concentration is added in the sodium-acetate buffer of 50mM pH 6.0,
Then with the Fe of 5mg3O4Magnetic particle mixing, by mixture vortex mixed, 4 DEG C of overnight incubations；It is washed twice with the PBS liquid of pH7.0
Then mixture is incubated at room temperature 30min in the Tris buffer of 50mM pH7.2；It is washed, is obtained with the PBS liquid of pH7.0 again
Biotin secondary antibody nanometer enzyme probe.Finally nanometer enzyme probe is distributed in the 5%BSA-PBS solution of 1mL.Use JEOL
2000FX 200kV transmission electron microscope (TEM) observes the partial size of nanometer enzyme probe, the size distribution display average diameter of MNP
For 200nm, it can be used for the building of nanometer enzyme sensor.
7, the preparation of nanometer enzyme sensor
The assembling of nanometer enzymatic nucleic acid chromatograph test strip (nanometer enzyme test peper): the test strips include sample pad, bonding pad, nitre
Acid cellulose film and absorption pad, wherein the nitrocellulose filter is equipped at least 1 detection line and 1 nature controlling line.
1. the preparation of sample pad and bonding pad: then the first sticking two-faced adhesive tape on bottom plate is pasted bonding pad, is then being combined
Sample pad, sample pad 2-4mm Chong Die with bonding pad are pasted on pad.The spy of biotin secondary antibody nano enzyme is wherein fixed on bonding pad
Needle.
2. FITC antibody and biotin antibody are diluted to optium concentration 1mg/mL with optimized buffer liquid respectively.It will dilute
FITC antibody-solutions be packed into BIODOT Film-cutting machine spray head 2, be fixed on the position away from NC film lower edge 1.1cm, diluted
Biotin two corresponding anti-solution is packed into BIODOT Film-cutting machine spray head 1, is fixed on the position away from NC film lower edge 1.6cm.Detection line (T
Line) it with the distance between nature controlling line (C line) is 4.5mm, it is sprayed on the T line and C line of NC film respectively by 1.0 μ L/cm.It will spray
It is spare after 37 DEG C of NC film good drying overnight.It is cut into the test paper of 3.8mm wide with cutting machine, the test paper cut is put into equipped with drying
In the packaging bag of agent.
3. above-mentioned sample pad, bonding pad, NC film and absorption pad with T line and C line are successively pasted on bottom plate, it is complete
At the assembling of test strips.
8, the detection of Enterobacter sakazakii
After completing PMA and LAMP operating procedure, detected with nanometer enzyme test peper.Detection includes two reaction steps
It is rapid: (1) hybridization reaction；(2) signal enhancing.
10 μ L LAMP-PCR amplified productions are taken, are mixed with 50 μ L reaction buffers (4 × SSC, 0.2m/m% Tween-20)
It is added drop-wise in the sample pad of above-mentioned nanometer enzymatic nucleic acid chromatograph test strip, after reacting 15min, is added dropwise on p-wire and nature controlling line afterwards
2 drop substrate buffer solutions (contain 20 × DAB and 20 × H2O2Commercial reagents), then reaction 5min observes by the naked eye colour developing
Situation, interpretation result: negative reaction: nature controlling line colour developing, detection line do not develop the color；Positive reaction: nature controlling line, detection line develop the color；
Failure reaction: if nature controlling line does not develop the color, failure or test strips failure are detected.
For quantitative measurment, the optics of the portable strip reader record strip band combined with band reader software is used
Intensity calculates the content of Enterobacter sakazakii in sample to be tested according to optical strength.
In order to verify accuracy of the biosensor established by the present invention in actual sample detection process, the present invention couple
Certain density Enterobacter sakazakii carries out mark-on reclaims verifying.Pass through standard culture and bacterium from local supermarket purchase baby milk powder
It is negative to fall counting method detection Enterobacter sakazakii.Then by Enterobacter sakazakii with 10,103With 105In the concentration incorporation milk powder of CFU/mL
It mixes.Successively progress PMA processing, genome extraction, LAMP amplification, nucleic acid test strip colour developing, peak area reading, quantitative analysis,
The rate of recovery of Enterobacter sakazakii shows that the present invention establishes in the range of 105 ± 2.5% to 108.7 ± 6.4% as the result is shown
Sensor detecting method can be used for the detection (table 4) of actual sample.
4 nanometers of enzyme sensors of table detect milk powder Enterobacter sakazakii content
The optimization of 2 nano enzyme nucleic acid test strip of embodiment
Fe is synthesized according to hydro-thermal method3O4Magnetic particle and biotin secondary antibody (sheep anti-mouse igg) are then incubated for by magnetic particle,
Prepare nanometer enzyme probe.It is utilized respectively the T line and the scribing line of C line position of FITC antibody and biotin antibody on NC film, after drying
It is assembled into nano enzyme nucleic acid test strip.It by comparing include the performance of membrane material to improve the sensitivity of nanometer enzyme sensor,
The concentration of detection zone FITC antibody, the amount of nanometer enzyme probe, reaction time are systematically analyzed.As a result it proves to use
The performance of the nanometer enzyme sensor of Millipore135S nitrocellulose filter is more preferable (Fig. 2A).Using 1mg/mL FITC antibody and
1mg/mL sheep anti-mouse igg, the signal peak area highest (Fig. 2 B) of acquisition.In addition, the amount of nanometer enzyme probe influences nanometer enzyme probe
Hybridization efficiency between sample, and the use of the nanometer enzyme probe of 10 μ L is optimal volume (Fig. 2 C).Test strips chromogenic reaction
Time is not less than 15min (Fig. 2 D).
The performance detection of 3 nanometers of enzyme sensors of embodiment
The principle of this nanometer of enzyme sensor is as follows: firstly, handling sample (step 1) with PMA.Wear to the PMA property of can choose
The cell membrane of saturating dead cell damage in conjunction with intracellular DNA, and makes it cannot be used for subsequent LAMP and expands, but if
When being the complete cell membrane of living cells, PMA cannot enter cell.Then, many BIO- are generated in a short time using LAMP
Duplex DNA (the step 2) connected with FITC-.In the presence of target substance ompA specific sequence, is identified and expanded by four kinds of primers
Increase.Third is the visualization interpretation (step 3) of nanometer enzymatic nucleic acid test paper.By physical absorption by FITC antibody and sheep anti-mouse igg
It is fixed on nitrocellulose filter, to be respectively formed detection zone (TL) and quality control region (CL).If sample is positive, process
LAMP amplification, 5 ' end biotin labelings of target substance, 3 ' end marked with FITC, sample solution again with nanometer enzyme probe knot
It closes.Then, the FITC antibody hybridization of conjugate and detection zone.Conjugate is formed by continue along band migration and pass through nano enzyme
Probe and sheep anti-mouse igg reaction, develop the color in quality control region.By DAB/H2O2When zymolyte is applied to detection zone and quality control region, nanometer
Enzyme and DAB/H2O2Enzymatic reaction between zymolyte will generate color reaction to enhance visual effect.The rugged intestines of slope living are being not present
In the case where bacillus, only quality control region develops the color.
The performance of nanometer enzyme sensor is assessed by the following aspects.First, reproducibility is in evaluation biosensor
Have great importance.By using the reproducibility of work Enterobacter sakazakii (Fig. 3 A) the test biosensor of 100CFU/mL, altogether
Test five times.The corresponding RSD value of optic response is 1.5%, shows that this nanometer of enzyme sensor has excellent reproducibility.Due to sample
The product always mixture of different bacterium type in practice, it is intended to make detection method that there is higher specificity.In order to comment
The specificity of valence nanometer enzyme sensor, using distilled water, the DNA of the dead bacterium of Enterobacter sakazakii, the DNA of Enterobacter sakazakii viable bacteria and its
He is Enterobacter bacteria bacterial strain DNA and other non-Enterobacter bacteria bacterial strain DNA.As a result as shown in Figure 3B, false sun is not observed
Property.The response holding of non-Enterobacter sakazakii and the dead bacterium of Enterobacter sakazakii is low as background signal, shows that non-specific adsorption exists
Enterobacter sakazakii living stems are had no significant effect to reaction system under the experiment condition.Early period by using LAMP amplification also into
One step improves the high specific of nanometer enzyme sensor.Stability (service life) is studied, is surveyed after storing 1-5 weeks at room temperature
Try the performance of nanometer enzyme sensor.As a result as shown in Figure 3 C, nanometer enzyme sensor protects the reaction of 100CFU/mL Enterobacter sakazakii
It holds almost the same, shows that the nano enzyme sensor stability is good.
The verifying of 4 nano enzyme transducer sensitivity of embodiment
In order to evaluate the sensitivity of nanometer enzyme sensor, the rugged intestines of the slope containing various concentration are measured under optimum experimental condition
Bacillus viable bacteria (range from 0 to 105CFU/mL sample solution) then measures the absorption peak area of TL line, three parallel laboratory tests.
It is being minimal in maximum magnitude, the gained figure to the response of Enterobacter sakazakii concentration is linear, and dependent equation is peak face
Product=18854lg Enterobacter sakazakii concentration+3115.6, coefficient R2It is 0.9818, is suitble to quantitative detection.Wherein, the rugged intestines of slope
Bacillus concentration unit is CFU/mL.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii
<130> KHP171113515.5
<160> 4
<170> PatentIn version 3.3
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<211> 19
<212> DNA
<213>artificial sequence
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tggtcccagt tccacgata 19
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<211> 19
<212> DNA
<213>artificial sequence
<400> 2
acgccctgag ctttgaaag 19
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<211> 42
<212> DNA
<213>artificial sequence
<400> 3
taacctggta accaccgaac gctattccta acgacggtcc ga 42
<210> 4
<211> 42
<212> DNA
<213>artificial sequence
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gtacgttggc ttcgaaatgg gccagtgtcg cctttatacg gc 42
Claims (10)
1. the LAMP primer group for detecting Enterobacter sakazakii (Enterobacter Sakazakii) characterized by comprising
Outside forward primer F3:5 '-TGGTCCCAGTTCCACGATA-3；'
Outside reverse primer B3:5 '-ACGCCCTGAGCTTTGAAAG-3；'
Inside forward primer FIP:5 '-TAACCTGGTAACCACCGAACGCTATTCCTAACGACGGTCCGA-3；'
Inside reverse primer BIP:5 '-GTACGTTGGCTTCGAAATGGGCCAGTGTCGCCTTTATACGGC-3；'
Wherein, 5 ' the end label biotins of primers F IP, 5 ' the end mark fluorescent element FITC of primer BIP.
2. for the method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii of non-diagnostic purposes, feature exists
In, comprising the following steps:
S1, sample to be tested DNA is extracted, using DNA as template, carries out LAMP-PCR expansion using LAMP primer group described in claim 1
Increase reaction；
S2, the LAMP-PCR amplified production for taking 10 μ L step S1, are added drop-wise to a nanometer enzymatic nucleic acid after mixing with 50 μ L reaction buffers
In the sample pad of chromatograph test strip, after 5-20min, 2 drop substrate buffer solutions are added dropwise on p-wire and nature controlling line, after reacting 5min
Observe by the naked eye colour developing situation, interpretation result: negative reaction: nature controlling line colour developing, detection line do not develop the color；Positive reaction: Quality Control
Line, detection line develop the color；Failure reaction: if nature controlling line does not develop the color, failure or test strips failure are detected；
For quantitative measurment, the optics using the portable strip reader record strip band combined with band reader software is strong
Degree, the content of Enterobacter sakazakii in sample to be tested is calculated according to optical strength；
Wherein, reaction buffer described in step S2 are as follows: 4 × SSC, 0.2m/m% Tween-20；Contain in the substrate buffer solution
20 × DAB and 20 × H2O2。
3. according to the method described in claim 2, it is characterized in that, in step S1 LAMP-PCR amplified reaction system are as follows: 1 ×
Bst Thermal Buffer, 0.6M Betaine, 0.5mM dNTPs solution, 1.6 μM of FIP, 1.6 μM of BIP, 0.2 μM of F3,
0.2 μM of B3,3.6mM MgSO4, 8U Bst archaeal dna polymerase large fragment, 2 μ L templates, ddH2O complements to 25 μ L；
LAMP-PCR response procedures are as follows: 65 DEG C of 30min, then 85 DEG C of 3min.
4. according to the method in claim 2 or 3, which is characterized in that further include being carried out to sample to be tested before step S1
The step of nitrine propidium iodide processing, specifically: final concentration is added in the sample to be tested for containing viable bacteria or hot inactivation of bacterial to 1 μ L
It for the nitrine propidium iodide of 10 μ g/mL, is protected from light and is incubated for 5min, then using 500W halogen light source by sample exposure 5min, by sample
Quality control is remotely from light source 20cm and is horizontally arranged on ice, shakes sample cell every 30s to keep to the uniform of light source
Exposure；Then DNA extraction is carried out.
5. according to the method described in claim 2, it is characterized in that, the preparation method packet of the nanometer enzymatic nucleic acid chromatograph test strip
Include following steps:
1)Fe3O4The preparation of magnetic particle；
2) preparation of nanometer enzyme probe: by Fe3O4Magnetic particle is incubated for biotin secondary antibody, obtains biotin secondary antibody nano enzyme
Probe；
3) assembling of nanometer enzymatic nucleic acid chromatograph test strip: the test strips include sample pad, bonding pad, nitrocellulose filter and suction
Receive pad, wherein the nitrocellulose filter is equipped at least 1 detection line and 1 nature controlling line；Life is fixed on the bonding pad
Object element secondary antibody nanometer enzyme probe；
1. marking detection line and nature controlling line on nitrocellulose filter with FITC antibody and biotin antibody respectively, dry；
2. above-mentioned sample pad, bonding pad, the nitrocellulose filter with detection line and nature controlling line and absorption pad are successively pasted
On bottom plate, the assembling of test strips is completed.
6. according to the method described in claim 5, it is characterized in that, the preparation method of the nanometer enzymatic nucleic acid chromatograph test strip walks
Rapid 1) middle utilization hydro-thermal method synthesizes Fe3O4Magnetic particle, specifically: by 0.6-0.8g FeCl3·6H2O is dissolved in 20mL ethylene glycol
In, 1.5-2.0g sodium acetate is then added, stirs 30-40min, is then sealed in autoclave, 200 DEG C of heating 16-18h；Magnetic
Granular product ethanol washing, and it is dry at 60 DEG C；Dichloroethanes and each 5-8mg of n-hydroxysuccinimide are passed through into vortex
It is dissolved in 1mL deionized water, mixed liquor is made, then 5-8mg magnetic particle is added in mixed liquor, is incubated for 30- at room temperature
Then 40min collects magnetic particle with magnet, with milli-Q water to get Fe3O4Magnetic particle.
7. according to the method described in claim 6, it is characterized in that, the preparation method of the nanometer enzymatic nucleic acid chromatograph test strip
In, step 2) specifically: the biotin secondary antibody of 100 μ g/mL of concentration is added in the sodium-acetate buffer of 50mM pH 6.0, so
Afterwards with the Fe of 5-8mg3O4Magnetic particle mixing, by mixture vortex mixed, 4 DEG C of overnight incubations；Mixing is washed with the PBS liquid of pH7.0
Then object is incubated at room temperature 30-40min in the Tris buffer of 50mM pH7.2；It is washed with the PBS liquid of pH7.0 again to get arriving
Biotin secondary antibody nanometer enzyme probe.
8. according to the method described in claim 5, it is characterized in that, the preparation method of the nanometer enzymatic nucleic acid chromatograph test strip walks
It is rapid 2) and 3) described in biotin secondary antibody be sheep anti-mouse igg；Nitrocellulose filter described in step 3) is
Millipore135S。
9. according to the described in any item methods of claim 5-8, which is characterized in that the system of the nanometer enzymatic nucleic acid chromatograph test strip
Detection line is located on the position away from nitrocellulose filter lower edge 1.1cm in Preparation Method step 3), and nature controlling line is located at away from nitric acid fibre
On the position for tieing up plain film lower edge 1.6cm；The distance between detection line and nature controlling line are 4.5mm, resist FITC by 1.0 μ L/cm
Body and biotin antibody are sprayed on respectively in the detection line and nature controlling line of nitrocellulose filter, are wherein wrapped on detection line and nature controlling line
The antibody concentration of quilt is 0.5-2mg/mL.
10. the kit containing LAMP primer group described in claim 1.
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