CN107385019A - Method based on biosensor technique detection Listeria Monocytogenes - Google Patents
Method based on biosensor technique detection Listeria Monocytogenes Download PDFInfo
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Abstract
The present invention relates to a kind of method based on nucleic acid chromatography biosensor technique detection Listeria Monocytogenes, according to the virulence gene hlyA of Listeria Monocytogenes, 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 Listeria Monocytogenes 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-cuts of Listeria Monocytogenes 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 Listeria Monocytogenes.
Background technology
Listeria Monocytogenes (Listeria monocytogenes) abbreviation Listeria monocytogenes, it is a kind of
The pathogen of zoonosis.It can cause Lee Salmonella disease of people and animals, and septicemia, meningitis and monokaryon are mainly shown as after infection
Cytosis.The bacterium is gram-positive short, and size is about 1.0-2.0 μm of 0.5 μ m (1.0-2.0), straight or slightly curved, two
Blunt circle is held, is often arranged in V-shape, occasionally there is spherical, double spherical, amphimicrobian, without brood cell.It is widely present in nature, in soil
With the presence of the bacterium in earth, surface water, sewage, waste water, plant, greenfeed, rotten dish, so animal is easy to eat the bacterium,
And propagated by fecal-oral route.It is reported that the carrying rate for singly increasing Lee Salmonella in healthy human faecal mass is 0.6-16%, the people for having 70%
It can carry disease germs in short term, 4-8% aquatic products, 5-10% milk and products thereof, more than 30% meat products and more than 15% poultry
Polluted by the bacterium.People mainly by eat soft cheese, the chicken not heated fully, the hot dog not heated again, fresh milk, bar
Family name's sterile milk, ice cream, raw steak, mutton chop, slaw, celery, tomato, French pie, frozen pork tongue etc. and infect, about
The case for accounting for 85-90% is as caused by contaminated food.
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 monocyte increasing is established
Raw listeria spp is reliable, quick sensor detecting method, the method that can particularly identify life or death bacterium, in daily prison
Control, market examination etc. are significant.
The content of the invention
It is an object of the invention to provide one kind based on nucleic acid chromatography biosensor technique detection monocyte hyperplasia Liszt
The method of Salmonella.
Present inventive concept is as follows：A kind of quick and super sensitivity detection method of Listeria Monocytogenes is developed,
The method that life or death bacterium can particularly be identified, foundation are based on nitrine propidium iodide (PMA), loop-mediated isothermal amplification (LAMP)
With the continuous cascade nanometer enzyme biologic sensor of the living stems of nanometer enzyme test peper.In LAMP reactions, fluorescein is used
(FITC) the hlyA genes of the primer measure Listeria Monocytogenes with biotin (BIO) modification of modification.By PMA
Combined with LAMP, the separation applied to life or death Listeria Monocytogenes.Then, prepared and be based on using nanometer enzyme probe
The immunochromatography bar (nano enzyme bar) of magnetic-particle is used to detect amplified signal, detects or is entered using bar graph-type readout by visual observation
The interpretation of row result is quantitative, for Site Detection Listeria Monocytogenes viable bacteria provide quickly, hypersensitive and easily
Instrument.
In order to realize the object of the invention, present invention firstly provides the LAMP for detecting Listeria Monocytogenes
Primer sets, including (SEQ ID NO:1-4)：
Outside forward primer F3：5’-AACAATGTATTAGTATACCACGG-3’；
Outside reverse primer B3：5’-GGAAGAACATCGGGTTGAT-3’；
Inner side forward primer FIP：5’-GGATTTCTTCTTTTTCTCCACAACGAGATGCAGTGACAAATGTG-3’；
Inner side reverse primer BIP：5’-TCCAAGTTGTGAATGCAATTTCGCCGAATTCGCTTTTACGAGA-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 0.6-0.8gFeCl3·6H2O 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 0.6gFeCl3·6H2O is dissolved in 20mL second
In glycol, 1.5g sodium acetates are then added, 30min is stirred vigorously, is then sealed in autoclave, 200 DEG C of heating 16h；Magnetic
Grain 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 vortex
It is dissolved in 1mL deionized waters, mixed liquor is made, then 5mg magnetic particle is added in mixed liquor, is incubated 30min at room temperature, so
Magnetic particle is collected with magnet afterwards, 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 Listeria Monocytogenes,
Comprise the following steps：
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 Listeria Monocytogenes 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 containing it is described be used for detect Listeria Monocytogenes LAMP primer group and/
Or the kit of the nanometer enzymatic 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 scene of Listeria Monocytogenes viable bacteria to examine
Disconnected test.
(4) one group of new LAMP primer is provided, the hlyA genes available for detection Listeria Monocytogenes.
(5) nanometer enzyme sensor to the Monitoring lower-cut of Listeria Monocytogenes up to 10CFU/mL.
Brief description of the drawings
Fig. 1 is Listeria Monocytogenes LAMP amplifications in the embodiment of the present invention 1；Wherein, M DNA
Marker, swimming lane 1-3 are Listeria Monocytogenes positive amplification, and 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-monocytic cells monocytogenes bacterial strain DNA cloning products, dead Listeria Monocytogenes bacterial strain
DNA cloning product, Listeria Monocytogenes bacterial strain DNA cloning product living；(C) sensor stability analysis result
Figure.It is from left to right the test result with a collection of sensor week about.
Fig. 4 is the linearity curve that Listeria Monocytogenes detect in the embodiment of the present invention 4：Peak area is with lg
The change of (Listeria Monocytogenes concentration).
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 Listeria Monocytogenes
1st, experiment material
Listeria Monocytogenes used are believed with non-monocytic cells monocytogenes bacterial strain in the present embodiment
Breath is shown in Table 1.
The information of Listeria Monocytogenes and non-monocytic cells monocytogenes used in table 1
The specificity of nanometer enzyme sensor is determined using Listeria Monocytogenes and other bacterium bacterial strains.
All bacterial strains are stored in -80 DEG C 20% (v/v) glycerite using preceding.Then it is incubated overnight in LB culture mediums
Activation.Pass through spectrophotometry Listeria Monocytogenes concentration.
2nd, Listeria Monocytogenes 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.Listeria Monocytogenes are separated using the bacterial genomes DNA extraction kit of New Ind μ stry companies
Genomic DNA, the genomic DNA of extraction carry out LAMP reactions immediately after preparation.Increase in the heat inactivation monocyte of PMA processing
In raw listeria spp sample, genomic DNA is not extracted, and sample can not be expanded using LAMP.And do not use
The life or death bacterium genomic DNA of PMA processing can be carried out expanding.It can be seen that the amplified band of dead bacterium is eliminated by PMA processing,
Only leave the amplified band of viable bacteria.Thus prove that PMA processing can effectively eliminate the DNA signals of dead bacterium.
4th, design of primers
The Listeria Monocytogenes gene hlyA announced according to document conserved region, pass through the flourish stock formula of Japan
Commercial firm ring mediation primer Photographing On-line software LAMP primer designing software primerexplorer V 4.0
(https://primerexplorer.jp/lamp4.0.0/index.html) for gene design primer, including draw outside 2
Thing F3, B3 and 2 inner primer FIP, BIP (table 2).In FIP 5 ' end mark biotins, BIP 5 ' end mark fluorescent elements
(FITC).Primer is synthesized by Shanghai Ying Weijieji biologies Co., Ltd, for follow-up screening and optimizing.
Primer sequence used in table 2LAMP
5th, the amplification of Listeria Monocytogenes genome
Listeria Monocytogenes 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 Listeria Monocytogenes genome as negative control group.The μ L of amplified production 5 are taken,
It is well mixed with 1 μ L sample-loading buffers, carries out electrophoresis with 2% Ago-Gel, allusion quotation occur in Listeria Monocytogenes
Type DNA cloning band (Fig. 1), non-monocytic cells monocytogenes bacterial strain do not produce any band, thus prove design
Primer pair Listeria Monocytogenes have 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 Listeria Monocytogenes
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 Listeria Monocytogenes 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 Listeria Monocytogenes carry out mark-on reclaims checking.From local supermarket, purchase ice cream passes through standard
Culture and colony counting method detection Listeria Monocytogenes are negative.Then by Listeria Monocytogenes with
10,103With 105Mixed in CFU/mL concentration incorporation ice cream.PMA processing, genome extraction, LAMP amplifications, core are carried out successively
Sour test strips colour developing, peak area is read, quantitative analysis, as a result shows the rate of recovery of Listeria Monocytogenes 98.1
In the range of ± 5.7% to 106.3 ± 7.3%, show that the sensor detecting method that the present invention establishes can be used for actual sample
Detection (table 4).
4 nanometers of enzyme sensor detection ice cream Listeria Monocytogenes 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 hlyA 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.Thin in the absence of monokaryon living
In the case of born of the same parents' monocytogenes, 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.Sensed by using 100CFU/mL work Listeria Monocytogenes (Fig. 3 A) test biology
The repeatability of device, test five times altogether.The corresponding RSD values of optic response are 1.5%, and it is excellent to show that this nanometer of enzyme sensor has
Repeatability.Due to the sample always mixture of different bacterium species in practice, it is intended to there is detection method higher
Specificity.In order to evaluate the specificity of nanometer enzyme sensor, using distilled water, the dead bacterium of Listeria Monocytogenes
DNA, the DNA of Listeria Monocytogenes viable bacteria and other Enterobacter bacteria bacterial strain DNA and other non-Enterobacters
Bacterium bacterial strain DNA.As a result as shown in Figure 3 B, false positive is not observed.Non-monocytic cells monocytogenes and monocyte
The response holding of the dead bacterium of monocytogenes is low as background signal, shows that non-specific adsorption is right under the experiment condition
Reaction system has no significant effect Listeria Monocytogenes living stems.Early stage also enters one by using LAMP amplifications
Step improves the high specific of nanometer enzyme sensor.Studied for stability (life-span), tested after storing 1-5 weeks at room temperature
The performance of nanometer enzyme sensor.As a result as shown in Figure 3 C, nanometer enzyme sensor is to 100CFU/mL monocyte hyperplasia Listerias
The reaction of bacterium keeps almost identical, shows 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, it is thin that the monokaryon containing various concentrations is measured under optimum experimental condition
(scope is from 0 to 10 for born of the same parents' monocytogenes viable bacteria5CFU/mL sample solution), the absorption peak area of TL lines is then measured,
Three parallel laboratory tests.It is being minimal in maximum magnitude, the gained figure of the response to Listeria Monocytogenes concentration is
It is linear, and dependent equation is peak area=21657lg Listeria Monocytogenes concentration+866.94, phase relation
Number R2For 0.996, it is adapted to quantitative detection.Wherein, Listeria Monocytogenes 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 biosensor technique detection Listeria Monocytogenes
<130> KHP171113513.3
<160> 4
<170> PatentIn version 3.3
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<213>Artificial sequence
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Claims (10)
1. the LAMP primer group for detecting Listeria Monocytogenes (Listeria monocytogenes), it is special
Sign is, including：
Outside forward primer F3：5’-AACAATGTATTAGTATACCACGG-3’；
Outside reverse primer B3：5’-GGAAGAACATCGGGTTGAT-3’；
Inner side forward primer FIP：5’-GGATTTCTTCTTTTTCTCCACAACGAGATGCAGTGACAAATGTG-3’；
Inner side reverse primer BIP：5’-TCCAAGTTGTGAATGCAATTTCGCCGAATTCGCTTTTACGAGA-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 Listeria Monocytogenes, it is characterised in that bag
Include 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 Listeria Monocytogenes 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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| CN107345961A (en) * | 2017-06-22 | 2017-11-14 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii |
| CN107419007A (en) * | 2017-06-22 | 2017-12-01 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection staphylococcus aureus |
| CN109402272A (en) * | 2018-08-24 | 2019-03-01 | 暨南大学 | Primer sets, kit and method based on intelligent constant-temperature amplification technique detection Listeria monocytogenes |
| CN110470841A (en) * | 2019-09-06 | 2019-11-19 | 齐鲁工业大学 | A kind of method of quantitative detection Listeria monocytogenes |
| WO2020136595A1 (en) | 2018-12-25 | 2020-07-02 | Tubitak | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method |
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| CN114686611A (en) * | 2022-04-24 | 2022-07-01 | 常州先趋医疗科技有限公司 | Primer group for detecting listeria monocytogenes and application thereof |
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