CN107345961A - 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 method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii, according to the virulence gene ompA of Enterobacter sakazakii, design loop-mediated isothermal amplification (LAMP) primer (SEQ ID NO:1 4), with reference to nano enzyme nucleic acid test strip, establish a kind of Enterobacter sakazakii detection method based on LAMP nanometer enzyme sensors.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 field, specifically, is related to a kind of based on nucleic acid chromatography bio-sensing
The method of technology for detection Enterobacter sakazakii.
Background technology
Enterobacter sakazakii (Cronobacter sakazakii category) is a kind of Gram-negative, is moved by peritrichous, be nonspore-bearing simultaneous
Property anaerobic bacteria.It is a kind of conditioned pathogen of Enterobacter, and the microbial food origin disease can cause baby (especially
Neonate) meningitis, enterocolitis and bacteremia, the death rate of reported cases reaches 20%-50%, and survivor often leaves sternly
The nervous system disease of weight.It is former to food (milk powder, chocolate, cereal preparation, potato, pasta etc.) processing factory in recent years
The research of Enterobacter sakazakii pollution rate is found in material, production environment and family, and Enterobacter sakazakii is widely distributed in nature.
Breaking out in event for a lot of Enterobacter sakazakiis finds that the infection of neonate 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 needs to increase before passing through
The steps such as bacterium, the separation of selective flat board, Biochemical Identification, determine that result needs 5-7 days from being sampled to, detection cycle length, behaviour
Make cumbersome, workload is big；Using the specificity of antigen-antibody reaction, bacterium is differentiated, the history of existing over half a century,
But the screening of microbial antibodies is very cumbersome, 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, also to carry out for microorganism
Quick determination method developed rapidly, but be to be not easy analysis result the shortcomings that molecular biology method.With existing
For the development of biotechnology, more faster than conventional method, more sensitive biology sensor new technology is in field of detection of food safety
Show up prominently.Biology sensor possesses the advantages that high specificity, high sensitivity, can shorten analysis with Simplified analysis detecting step
Time, it is often more important that make it possible on-line real-time measuremen, be easy to carry and field work, obtained in field of food safety
Faster development.In recent years, the development and application of numerous new materials, new technology is just turning into the focus of research, is biology sensor
Development be filled with new vitality, huge application potential is shown in microorganism detection field.Therefore Enterobacter sakazakii is established
Reliably, quick sensor detecting method, the method that can particularly identify life or death bacterium, in daily monitoring, market examination etc.
Aspect is significant.
The content of the invention
It is an object of the invention to provide a kind of method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii.
Present inventive concept is as follows：A kind of quick and super sensitivity detection method of Enterobacter sakazakii is developed, can particularly be reflected
The method for fixing viable bacteria, foundation are based on nitrine propidium iodide (PMA), loop-mediated isothermal amplification (LAMP) and nanometer enzyme test peper
Living stems continuous cascade nanometer enzyme biologic sensor.It is being modified using fluorescein (FITC) and raw in LAMP reactions
The ompA genes of the primer measure Enterobacter sakazakii of thing element (BIO) modification.PMA is combined with LAMP, applied to the rugged intestines of life or death slope
The separation of bacillus.Then, the immunochromatography bar (nano enzyme bar) based on magnetic-particle is prepared using nanometer enzyme probe to be used to detect
Amplified signal, detect by visual observation or carry out result interpretation or quantitative using bar graph-type readout, be Site Detection Enterobacter sakazakii
Viable bacteria provides quick, hypersensitive and easily instrument.
In order to realize the object of the 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’；
Inner side forward primer FIP：5’-TAACCTGGTAACCACCGAACGCTATTCCTAACGACGGTCCGA-3’；
Inner side reverse primer BIP：5’-GTACGTTGGCTTCGAAATGGGCCAGTGTCGCCTTTATACGGC-3’.
Wherein, primers F IP 5 ' end mark biotins, primer BIP 5 ' end mark fluorescent element FITC.
The present invention also provides a kind of nanometer enzymatic nucleic acid chromatograph test strip, and the preparation method of the test strips includes following step
Suddenly：
1)Fe3O4The preparation of magnetic particle；
2) preparation of nanometer enzyme probe (MNP)：By Fe3O4Magnetic particle is incubated with 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, pad, nitrocellulose filter
And absorption pad, wherein, the nitrocellulose filter is provided with least 1 detection line and 1 nature controlling line；It is fixed on the 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. by above-mentioned sample pad, pad, the nitrocellulose filter with detection line and nature controlling line and absorption pad successively
It is pasted onto on bottom plate (plastics lining board), completes the assembling of test strips.The structural representation of the test strips assembled is shown in Fig. 5.
Hydro-thermal method synthesis Fe is utilized in step 1)3O4Magnetic particle, it is specially：By 0.6-0.8g FeCl3·6H2O is dissolved in
In 20mL ethylene glycol, 1.5-2.0g sodium acetates are then added, 30-40min is stirred vigorously, is then sealed in autoclave, 200 DEG C
Heat 16-18h；Magnetic granular product is washed several times with ethanol, and is dried at 60 DEG C；Dichloroethanes and N- hydroxysuccinimidyls acyl is sub-
Each 5-8mg of amine is dissolved in 1mL deionized waters by being vortexed, and mixed liquor is made, and 5-8mg magnetic particle then is added into mixed liquor
In, 30-40min is incubated at room temperature, is then collected magnetic particle with magnet, is produced Fe with milli-Q water twice3O4Magnetic particle.
Step 2) is specially：The μ g/mL of concentration 100 biological antibody is added in 50mM pH 6.0 sodium-acetate buffer,
Then with 5-8mg Fe3O4Magnetic particle mixes, by mixture vortex mixed, 4 DEG C of overnight incubations；Two are washed with pH7.0 PBS liquid
Secondary mixture, then it is incubated at room temperature 30min in 50mM pH7.2 Tris buffer solutions；Washed again with pH7.0 PBS liquid, 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 cellulose membrane lower edge 1.6cm position.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 in the detection line and nature controlling line of nitrocellulose filter respectively.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.
The biotin antibody used in the present invention is purchased from Sigma companies, and goods number B7653, FITC antibody are 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：
Hydro-thermal method synthesis Fe is utilized in step 1)3O4Magnetic particle, it is specially：By 0.6g FeCl3·6H2O is dissolved in 20mL
In ethylene glycol, 1.5g sodium acetates are then added, 30min is stirred vigorously, is then sealed in autoclave, 200 DEG C of heating 16h；Magnetic
Granular product is washed several times with ethanol, and is dried at 60 DEG C；Dichloroethanes and each 5mg of n-hydroxysuccinimide are passed through into whirlpool
Rotation is dissolved in 1mL deionized waters, and mixed liquor is made, and is then added in mixed liquor 5mg magnetic particle, is incubated 30min at room temperature,
Then magnetic particle is collected with magnet, produces Fe with milli-Q water twice3O4Magnetic particle.
Step 2) is specially：The μ g/mL of concentration 100 biotin secondary antibody is added to 50mM pH6.0 sodium-acetate buffer
In, then with 5mg Fe3O4Magnetic particle mixes, by mixture vortex mixed, 4 DEG C of overnight incubations；Washed with pH7.0 PBS liquid
Mixture twice, then it is incubated at room temperature 30min in 50mM pH7.2 Tris buffer solutions；Washed again with pH7.0 PBS liquid,
Obtain biotin secondary antibody nanometer enzyme probe.Finally nanometer enzyme probe is distributed in 1mL 5%BSA-PBS solution.Use
JEOL 2000FX 200kV transmission electron microscopes (TEM) observe the particle diameter of nanometer enzyme probe, and MNP Size Distribution, which is shown, puts down
A diameter of 200nm, the structure available for nanometer enzyme sensor.
By sample pad, the 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 pasted onto on bottom plate successively, that is, complete the assembling of test strips.
The present invention also provides the method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii, including following step
Suddenly：
S1, extraction testing sample DNA, using DNA as template, it is anti-to carry out LAMP-PCR amplifications using above-mentioned LAMP primer group
Should；
S2, the LAMP-PCR amplified productions for taking 10 μ L steps S1, above-mentioned receive is added drop-wise to after being mixed 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
Control and 2 drop substrate buffer solutions are added dropwise on line, then colour developing situation, sentence read result are observed by the naked eye after reacting 5min：Negative reaction：
Nature controlling line develops the color, and detection line does 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 detection 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, the content of Enterobacter sakazakii in testing sample is calculated according to optical strength.
Wherein, reaction buffer is described in step S2：4 × SSC, 0.2m/m% Tween-20.
The 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 reactions is：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：65 DEG C of 30min, then 85 DEG C of 3min.
The step of nitrine propidium iodide is handled is carried out before step S1, in addition to testing sample, is specially：To 1 μ L
Final concentration of 10 μ g/mL nitrine propidium iodide is added in testing sample containing viable bacteria or hot inactivation of bacterial, lucifuge is incubated
5min, then sample cell is remotely from light source 20cm and level is put by sample exposure 5min using 500W halogen light sources
Put on ice, shake sample cell every 30s to keep the uniform exposure to light source；Then DNA extractions are carried out.
The present invention further provides be used to detect the LAMP primer group of Enterobacter sakazakii and/or the nano enzyme containing described
The kit of nucleic acid chromatograph test strip.
The present invention has advantages below：
(1) present invention develops the nanometer enzyme biologic sensor that PMA, LAMP are combined with nanometer enzyme test peper first.
(2) nanometer enzyme test peper is successfully used to distinguish living bacterial cells and dead bacterium cell first.
(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, the ompA genes available for detection Enterobacter sakazakii.
(5) nanometer enzyme sensor to the Monitoring lower-cut of Enterobacter sakazakii up to 10CFU/mL.
Brief description of the drawings
Fig. 1 is Enterobacter sakazakii LAMP amplifications 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 expands to be negative.
Fig. 2 is the optimization to biology sensor in the embodiment of the present invention 2；Wherein, (A) membrane material is to biology sensor peak face
Long-pending influence；(B) influence of the FITC antibody concentrations to biology sensor peak area in detection line；(C) dosage of nanometer enzyme probe
Influence of the volume to the peak area of biology sensor；(D) influence of the reaction time to the peak area of biology sensor.
Fig. 3 is performance of biosensor analysis result figure in the embodiment of the present invention 3；Wherein, (A) sensor reappearance is analyzed
Result figure.(B) sensor specificity analysis 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 analysis result figure.It is from left to right the survey with a collection of sensor week about
Test result.
Fig. 4 is the linearity curve that Enterobacter sakazakii detects in the embodiment of the present invention 4：(Enterobacter sakazakii is dense with lg for peak area
Degree) change.
Fig. 5 is the structural representation of nanometer enzymatic nucleic acid chromatograph test strip of the present invention.Wherein, 1- sample pads, 2- cellulose nitrates
Plain film, 3- pads, 4- absorption pads, 5- p-wires, 6- nature controlling lines, 7- bottom plates.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or the condition according to manufacturer's specification suggestion.
Method of the embodiment 1 based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii
1st, experiment material
Enterobacter sakazakii used is shown in Table 1 with non-Enterobacter sakazakii bacterial strain information in the present embodiment.
The information of Enterobacter sakazakii and non-Enterobacter sakazakii used in table 1
The specificity of nanometer enzyme sensor is determined using Enterobacter sakazakii and other bacterium bacterial strains.Will be all before use
Bacterial strain is stored in -80 DEG C 20% (v/v) glycerite.It is then incubated overnight activation in LB culture mediums.By dividing
Light photometry determines Enterobacter sakazakii concentration.
2nd, Enterobacter sakazakii genome extracts
Using the bacterial genomes DNA extraction kit of New Industry companies, comprise the following steps that：
(1) inoculum 1.5mL, 12000g centrifugation 1min is taken, exhausts supernatant as far as possible.
(2) 200 μ L solution As are added into bacterial precipitation, vibrates to thalline and fully suspends.
(3) 20 μ L10mg/mL Proteinase K is added into pipe, is fully mixed, centrifuge tube during which can be overturned and mix number,
Untill treatments of the sample is complete.It is that liquid is limpid and sticky to digest complete mark.
(4) 2000 μ L solution Bs are added into pipe, are fully mixed.Such as there is white precipitate, 75 DEG C of 15-30min can be placed in,
Precipitation can disappear, and not influence subsequent experimental.
(5) 220 μ L absolute ethyl alcohols are added into pipe, are fully mixed, now 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 centrifuges 1min, waste liquid, adsorption column is put into collecting pipe.
(7) 700 μ L rinsing liquids are added into adsorption column, 12000g centrifugation 1min, waste liquid, adsorption column are put into collecting pipe
In.
(8) 500 μ L rinsing liquids are added into adsorption column, 12000g centrifugation 1min, waste liquid, adsorption column are put into collecting pipe
In.
(9) 12000g centrifuges 2min, and adsorption column is placed in into 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 are added dropwise in adsorbed film center through 75
The eluent of DEG C water-bath preheating, room temperature place 2min, 12000g centrifugations 2min.
(11) centrifugation gained eluent is added in adsorption column, and room temperature places 2min, 12000g centrifugations 2min, you can obtain
The bacterial genomes DNA of high quality.
3rd, nitrine propidium iodide is handled
By 1 μ L viable bacterias or hot inactivation of bacterial, the final concentration of 10 μ g/mL of addition PMA.Sample lucifuge is incubated 5min, and
Mixing makes PMA penetrate into dead cell.Then 500W halogen light sources are used by sample exposure 5min.Sample cell is remotely from
At light source 20cm and lie in a horizontal plane on ice to avoid superheated.Sample cell is shaken per 30s to keep to the uniform sudden and violent of light source
Dew.The genomic DNA of Enterobacter sakazakii is separated using the bacterial genomes DNA extraction kit of New Ind μ stry companies, carried
The genomic DNA taken carries out LAMP reactions immediately after preparation.In the heat inactivation Enterobacter sakazakii sample of PMA processing, do not have
Genomic DNA is extracted, and sample can not be expanded using LAMP.And not using the life or death bacterium genomic DNA of PMA processing
It can be carried out expanding.It can be seen that eliminating the amplified band of dead bacterium by PMA processing, the amplified band of viable bacteria is only left.Thus
Prove that PMA processing can effectively eliminate the DNA signals of dead bacterium.
4th, design of primers
The Enterobacter sakazakii gene ompA announced according to document conserved region, drawn by the ring mediation of Japanese Rong Yan Co., Ltd.
(the https of thing Photographing On-line software LAMP primer designing software primerexplorer V 4.0://
Primerexplorer.jp/lamp4.0.0/index.html) for gene design primer, including 2 outer primers F3, B3 and 2
Bar inner primer FIP, BIP (table 2).It is plain (FITC) in FIP 5 ' end mark biotins, BIP 5 ' end mark fluorescents.Primer is by upper
Hai Yingweijieji biologies Co., Ltd synthesizes, for follow-up screening and optimizing.
Primer sequence used in table 2LAMP
5th, the amplification of Enterobacter sakazakii genome
Enterobacter sakazakii genome is expanded (table 3) using ring mediated isothermal amplification method.
Table 3LAMP reaction systems
LAMP response procedures are：30min is reacted at 65 DEG C, reacting 3min at 85 DEG C inactivates enzyme.This experiment is used and often managed
25 μ L system, wherein being not added with Enterobacter sakazakii genome as negative control group.The μ L of amplified production 5 are taken, with 1 μ L loading buffers
Liquid is well 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 produce any band, thus proves the primer pair Enterobacter sakazakii of design and has 100% specificity.
6th, 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 acetates are added, 30min is stirred vigorously, is then sealed in autoclave, 200 DEG C of heating 16h；Magnetic granular product ethanol
Washing several times, and is dried 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, 30min is incubated at room temperature, is then received with magnet
Collect magnetic particle, produce Fe with milli-Q water twice3O4Magnetic particle.
The μ g/mL of concentration 100 biotin secondary antibody (sheep anti-mouse igg) is added in 50mM pH 6.0 sodium-acetate buffer,
Then with 5mg Fe3O4Magnetic particle mixes, by mixture vortex mixed, 4 DEG C of overnight incubations；Washed twice with pH7.0 PBS liquid
Mixture, then it is incubated at room temperature 30min in 50mM pH7.2 Tris buffer solutions；Washed, obtained with pH7.0 PBS liquid again
Biotin secondary antibody nanometer enzyme probe.Finally nanometer enzyme probe is distributed in 1mL 5%BSA-PBS solution.Use JEOL
2000FX 200kV transmission electron microscopes (TEM) observe the particle diameter of nanometer enzyme probe, and MNP Size Distribution shows average diameter
For 200nm, the structure available for nanometer enzyme sensor.
7th, 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, pad, nitre
Acid cellulose film and absorption pad, wherein, the nitrocellulose filter is provided with least 1 detection line and 1 nature controlling line.
1. the preparation of sample pad and pad：The first sticking two-faced adhesive tape on bottom plate, then pastes pad, is then combining
Sample pad, sample pad 2-4mm overlapping with pad are pasted on pad.The spy of biotin secondary antibody nano enzyme is wherein fixed with pad
Pin.
2. FITC antibody and biotin antibody are diluted to optium concentration 1mg/mL with optimized buffer liquid respectively.It will dilute
FITC antibody-solutions load BIODOT Film-cutting machines shower nozzle 2, be fixed on the position away from NC film lower edges 1.1cm, diluted
Biotin two corresponding anti-solution loads BIODOT Film-cutting machines shower nozzle 1, is fixed on the position away from NC film lower edges 1.6cm.Detection line (T
Line) with the distance between nature controlling line (C lines) it is 4.5mm, it is sprayed on respectively on the T lines and C lines of NC films by 1.0 μ L/cm.It will spray
It is standby after good 37 DEG C of drying overnight of NC films.The wide test paper of 3.8mm is cut into cutting machine, the test paper cut is put into equipped with drying
In the packaging bag of agent.
3. above-mentioned sample pad, pad, NC films and absorption pad with T lines and C lines are pasted onto on bottom plate successively, it is complete
Into the assembling of test strips.
8th, the detection of Enterobacter sakazakii
After PMA and LAMP operating procedures are completed, detected with nanometer enzyme test peper.Detection walks comprising two reactions
Suddenly：(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), reaction and then observes by the naked eye colour developing at 5min
Situation, sentence read result：Negative reaction：Nature controlling line develops the color, and detection line does 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, detection failure or test strips failure.
For quantitative measurment, the optics of the portable strip reader record strip band combined with band reader software is used
Intensity, the content of Enterobacter sakazakii in testing sample is calculated according to optical strength.
It is of the invention right in order to verify accuracy of the biology sensor of the invention established in actual sample detection process
Certain density Enterobacter sakazakii carries out mark-on reclaims checking.From local supermarket, purchase baby milk powder passes through standard culture and bacterium
It is negative to fall counting method detection Enterobacter sakazakii.Then by Enterobacter sakazakii with 10,103With 105In CFU/mL concentration incorporation milk powder
Mix.PMA processing, genome extraction, LAMP amplifications, nucleic acid test strip colour developing are carried out successively, peak area is read, quantitative analysis,
As a result the rate of recovery of Enterobacter sakazakii is shown in the range of 105 ± 2.5% to 108.7 ± 6.4%, shows that the present invention establishes
Sensor detecting method can be used for the detection (table 4) of actual sample.
4 nanometers of enzyme sensor detection milk powder Enterobacter sakazakii contents of table
The optimization of the nano enzyme nucleic acid test strip of embodiment 2
Fe is synthesized according to hydro-thermal method3O4Magnetic particle, then magnetic particle and biotin secondary antibody (sheep anti-mouse igg) are incubated,
Prepare nanometer enzyme probe.The T lines and the line of C line positions of FITC antibody and biotin antibody on NC films are utilized respectively, after drying
It is assembled into nano enzyme nucleic acid test strip.In order to improve the sensitivity of nanometer enzyme sensor, by comparing the performance including membrane material,
The concentration of detection zone FITC antibody, the amount of nanometer enzyme probe, reaction time are systematically analyzed.As a result prove to use
The performance of the nanometer enzyme sensor of Millipore135S nitrocellulose filters is more preferable (Fig. 2A).Using 1mg/mL FITC antibody and
1mg/mL sheep anti-mouse iggs, 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 10 μ L nanometer enzyme probe 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：First, sample (step 1) is handled with PMA.PMA can optionally be worn
The cell membrane of saturating dead cell damage, is combined with intracellular DNA, and it is cannot be used for subsequent LAMP and expanded, but if
When being the complete cell membrane of living cells, PMA cannot enter cell.Then, many BIO- are produced in a short time using LAMP
Duplex DNA (the steps 2) connected with FITC-.In the presence of target substance ompA specific sequences, identified and expanded by four kinds of primers
Increase.3rd 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 form detection zone (TL) and quality control region (CL) respectively.If sample is positive, pass through
LAMP expand, 5 ' end biotin labelings of target substance, 3 ' end marked with FITC, sample solution again with nanometer enzyme probe knot
Close.Then, the FITC antibody hybridizations of conjugate and detection zone.The conjugate formed continues along band migration and passes 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 is by color reaction is produced to strengthen visual effect.In the absence of the rugged intestines of slope living
In the case of bacillus, only quality control region develops the color.
The performance of nanometer enzyme sensor is assessed by the following aspects.First, reappearance is in biology sensor is evaluated
Have great importance.It is common by using the repeatability of 100CFU/mL work Enterobacter sakazakii (Fig. 3 A) test biology sensor
Test five times.The corresponding RSD values of optic response are 1.5%, show that this nanometer of enzyme sensor has excellent repeatability.Due to sample
The product always mixture of different bacterium species in practice, it is intended to make detection method that there is higher specificity.In order to comment
The specificity of valency 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 3 B, false sun is not observed
Property.The response holding of the non-dead bacterium of Enterobacter sakazakii and Enterobacter sakazakii is low as background signal, shows that non-specific adsorption exists
Enterobacter sakazakii living stems are had no significant effect under the experiment condition to reaction system.Early stage also enters by using LAMP amplifications
One step improves the high specific of nanometer enzyme sensor.Studied for stability (life-span), 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, reaction of the nanometer enzyme sensor to 100CFU/mL Enterobacter sakazakiis is protected
Hold almost identical, show that the nano enzyme sensor stability is good.
The nano enzyme transducer sensitivity of embodiment 4 is verified
In order to evaluate the sensitivity of nanometer enzyme sensor, the rugged intestines of the slope containing various concentrations are measured under optimum experimental condition
(scope is from 0 to 10 for bacillus viable bacteria5CFU/mL sample solution), the absorption peak area of TL lines, three parallel laboratory tests are then measured.
It is being minimal in maximum magnitude, the gained figure of the response to Enterobacter sakazakii concentration is linear, and dependent equation is peak face
Product=18854lg Enterobacter sakazakiis concentration+3115.6, coefficient R2For 0.9818, it is adapted 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 improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present 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
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<213>Artificial sequence
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taacctggta accaccgaac gctattccta acgacggtcc ga 42
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Claims (10)
1. the LAMP primer group for detecting Enterobacter sakazakii (Enterobacter Sakazakii), it is characterised in that including：
Outside forward primer F3：5’-TGGTCCCAGTTCCACGATA-3’；
Outside reverse primer B3：5’-ACGCCCTGAGCTTTGAAAG-3’；
Inner side forward primer FIP：5’-TAACCTGGTAACCACCGAACGCTATTCCTAACGACGGTCCGA-3’；
Inner side reverse primer BIP：5’-GTACGTTGGCTTCGAAATGGGCCAGTGTCGCCTTTATACGGC-3’；
Wherein, primers F IP 5 ' end mark biotins, primer BIP 5 ' end mark fluorescent element FITC.
2. a kind of nanometer enzymatic nucleic acid chromatograph test strip, it is characterised in that the preparation method of the test strips comprises the following steps：
1)Fe3O4The preparation of magnetic particle；
2) preparation of nanometer enzyme probe：By Fe3O4Magnetic particle is incubated with 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, pad, nitrocellulose filter and suction
Pad is received, wherein, the nitrocellulose filter is provided with least 1 detection line and 1 nature controlling line；Life is fixed with the pad
Thing 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, pad, the nitrocellulose filter with detection line and nature controlling line and absorption pad are pasted successively
On bottom plate, the assembling of test strips is completed.
3. test strips according to claim 2, it is characterised in that hydro-thermal method synthesis Fe is utilized in step 1)3O4Magnetic particle,
Specially：By 0.6-0.8g FeCl3·6H2O is dissolved in 20mL ethylene glycol, then adds 1.5-2.0g sodium acetates, stirs 30-
40min, then it is sealed in autoclave, 200 DEG C of heating 16-18h；Magnetic granular product is washed with ethanol, and is dried at 60 DEG C；
Dichloroethanes and each 5-8mg of n-hydroxysuccinimide are dissolved in 1mL deionized waters by being vortexed, mixed liquor is made, so
5-8mg magnetic particle is added in mixed liquor afterwards, is incubated 30-40min at room temperature, then magnetic particle is collected with magnet, uses ultra-pure water
Washing, produces Fe3O4Magnetic particle.
4. test strips according to claim 3, it is characterised in that step 2) is specially：By the μ g/mL of concentration 100 biology
Plain secondary antibody is added in 50mM pH 6.0 sodium-acetate buffer, then with 5-8mg Fe3O4Magnetic particle mixes, by mixture whirlpool
Rotation mixing, 4 DEG C of overnight incubations；Mixture is washed with pH7.0 PBS liquid, then the room in 50mM pH7.2 Tris buffer solutions
Temperature is incubated 30-40min；Washed again with pH7.0 PBS liquid, that is, obtain biotin secondary antibody nanometer enzyme probe.
5. test strips according to claim 2, it is characterised in that step 2) and 3) described in biotin secondary antibody be goat-anti
Mouse IgG；Nitrocellulose filter described in step 3) is Millipore135S.
6. according to the test strips described in claim any one of 1-5, it is characterised in that detection line is located at fine away from nitric acid in step 3)
On the position for tieing up plain film lower edge 1.1cm, nature controlling line is located on the position away from nitrocellulose filter lower edge 1.6cm；Detection line
The distance between nature controlling line is 4.5mm, and FITC antibody and biotin antibody are sprayed on into cellulose nitrate respectively by 1.0 μ L/cm
In the detection line and nature controlling line of plain film, wherein coated antibody concentration is 0.5-2mg/mL in detection line and nature controlling line.
7. the method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii, it is characterised in that comprise the following steps：
S1, extraction testing sample DNA, using DNA as template, LAMP-PCR expansions are carried out using LAMP primer group described in claim 1
Increase reaction；
S2, the LAMP-PCR amplified productions for taking 10 μ L steps S1, claim 2-6 is added drop-wise to after being mixed with 50 μ L reaction buffers
In the sample pad of any one test strips, after 5-20min, 2 drop substrate buffer solutions, reaction are added dropwise on p-wire and nature controlling line
Colour developing situation, sentence read result are observed by the naked eye after 5min：Negative reaction：Nature controlling line develops the color, and detection line does not develop the color；It is positive anti-
Should：Nature controlling line, detection line develop the color；Failure reaction：If nature controlling line does not develop the color, detection failure or test strips failure；
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 testing sample is calculated according to optical strength；
Wherein, reaction buffer is described in step S2：4 × SSC, 0.2m/m% Tween-20；Contain in the substrate buffer solution
20 × DAB and 20 × H2O2。
8. according to the method for claim 7, it is characterised in that the system of LAMP-PCR amplified reactions is in step S1：1×
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 fragments, 2 μ L templates, ddH2O complements to 25 μ L；
LAMP-PCR response procedures are：65 DEG C of 30min, then 85 DEG C of 3min.
9. the method according to claim 7 or 8, it is characterised in that carried out before step S1, in addition to testing sample
The step of nitrine propidium iodide processing, it is specially：Final concentration is added in the testing sample for containing viable bacteria or hot inactivation of bacterial to 1 μ L
For 10 μ g/mL nitrine propidium iodide, lucifuge is incubated 5min, then using 500W halogen light sources by sample exposure 5min, by sample
QC is remotely from light source 20cm and lain in a horizontal plane on ice, shakes sample cell every 30s to keep to the uniform of light source
Exposure；Then DNA extractions are carried out.
10. the kit containing any one of LAMP primer group and/or claim the 2-6 test strips described in claim 1.
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