CN107340389B - Method based on nucleic acid chromatography biosensor technique detection salmonella - Google Patents
Method based on nucleic acid chromatography biosensor technique detection salmonella Download PDFInfo
- Publication number
- CN107340389B CN107340389B CN201710481882.1A CN201710481882A CN107340389B CN 107340389 B CN107340389 B CN 107340389B CN 201710481882 A CN201710481882 A CN 201710481882A CN 107340389 B CN107340389 B CN 107340389B
- Authority
- CN
- China
- Prior art keywords
- salmonella
- nucleic acid
- sample
- nanometer
- lamp
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Nanotechnology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of methods based on nucleic acid chromatography biosensor technique detection salmonella, according to the virulence gene invA of salmonella, 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 Detection Methods of Salmonella 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 salmonella 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 Detection of Salmonella.
Background technique
Salmonella (Salmonella spp.), the brevibacterium (thinner than Escherichia coli) of Gram-negative, both ends blunt circle,
(0.7~1.5 μm) × (2~5 μm) are dispersed in, no pod membrane and gemma, are had whole body flagellum, can be moved, most of to have pili.It is husky
Door Salmonella disease is one of the zoonosis being of great significance on public hygienics, cause of disease Salmonella enteric bacteria
Section, including those cause to poison by food, and lead to the bacterium of gastroenteritis, Typhoid and paratyphoid.They can also feel in addition to it can infect people
Contaminating many animals includes mammality, bird, reptiles, fish, amphibian animal and insect.It can be in asymptomatic carrier state after human poultry infection,
It can also appear as the lethal disease of clinical symptoms, it may aggravate morbid state or the death rate, or reduce the Breeding productivity of animal.
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, there has been going through for over half a century
History, but the screening of microbial antibodies is very complicated, and final detection specificity is not high；Molecular Biological Detection technology
It constantly improve and develops, overcome the problems such as traditional detection method experimental implementation is cumbersome, time-consuming, but also being opened for microorganism
The rapid detection method of exhibition is rapidly developed, but is to be not easy to analyze result the shortcomings that molecular biology method.With
The development of modern biotechnology, this new technology more faster than conventional method, more sensitive of biosensor are examined in food safety
Show up prominently in survey field.Biosensor has the advantages that high specificity, high sensitivity, can simplify analysis detection step, contracting
Short analysis time, it is often more important that make it possible on-line real-time measuremen, easy to carry and field work, in field of food safety
Faster development is obtained.In recent years, the development and application of numerous new materials, new technology is just having become a hot topic of research passes for biology
The development of sensor is filled with new vitality, shows huge application potential in microorganism detection field.Therefore Salmonella is established
Bacterium is reliable, quick sensor detecting method, the method that can especially identify life or death bacterium, in daily monitoring, market screening
Etc. be 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 salmonella.
Present inventive concept is as follows: developing a kind of quick and super sensitivity detection method of salmonella, can especially identify
The method of life or death bacterium is established and is based on nitrine propidium iodide (PMA), loop-mediated isothermal amplification (LAMP) and nanometer enzyme test peper
The continuous cascade nanometer enzyme biologic sensor of living stems.In LAMP reaction, using fluorescein (FITC) modification and biological
The invA gene of the primer measurement salmonella of plain (BIO) modification.It combines PMA with LAMP, applied to life or death salmonella
Separation.Then, using nanometer enzyme probe immunochromatography item (nano enzyme item) of the preparation based on magnetic-particle for detecting amplification letter
Number, it detects or using bar graph-type readout progress result interpretation or quantitatively, is provided for on-site test salmonella viable bacteria by visual observation
Quickly, hypersensitive and convenient and fast tool.
In order to achieve the object of the present invention, present invention firstly provides for detecting salmonella (Salmonella spp.)
LAMP primer group, including (SEQ ID NO:1-4):
Outside forward primer F3:5 '-GGATGACTCGCCATGGTATG-3 '；
Outside reverse primer B3:5 '-TTGTTCAACAGCTGCGTCA-3 '；And
Inside forward primer FIP:5 '-CTGGGCGACAAGACCATCACCATTTGTCCTCCGCCCTGT-3 '；
Inside reverse primer BIP:5 '-TCCCCGCATTGTTGATTGCGATCCGCCCCATATTATCCGTA-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, B7653, and FITC antibody is purchased from Sigma, F5636.Biology
Plain 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 salmonella, comprising the following steps:
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 salmonella 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 (PMA) 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 and incubates
5min is educated, then sample cell is remotely from sample exposure 5min at light source 20cm to simultaneously horizontal using 500W halogen light source
It is put on ice for, 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 salmonella and/or the nano enzyme core
The kit of sour 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 salmonella viable bacteria.
(4) one group of new LAMP primer is provided, can be used for detecting the invA gene of salmonella.
(5) nanometer enzyme sensor is to the Monitoring lower-cut of salmonella up to 10CFU/mL.
Detailed description of the invention
Fig. 1 is salmonella LAMP amplification in the embodiment of the present invention 1；Wherein, M is DNA Marker, and swimming lane 1-3 is
Salmonella positive amplification, swimming lane 4 are 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 nonsalmonella bacterial strain DNA cloning products, dead Salmonella strains DNA cloning product, Salmonella strains living
DNA cloning product；(C) sensor stability analyzes result figure: the test knot for same a collection of sensor every other week from left to right
Fruit.
Fig. 4 is the linearity curve of Salmeterol fluticasone propionate in the embodiment of the present invention 4: peak area is with lg's (salmonella concentration)
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 salmonella
1, experimental material
Salmonella used and nonsalmonella bacterial strain information are shown in Table 1 in the present embodiment.
The information of salmonella used in table 1 and nonsalmonella
The specificity of nanometer enzyme sensor is measured using salmonella and other bacterium bacterial strains.It is preceding by all bacterium using
Strain is stored in -80 DEG C 20% (v/v) glycerite.It is then incubated overnight activation in LB culture medium.Pass through light splitting
Photometry measures salmonella concentration.
2, salmonella gene group is extracted
Using the bacterial genomes DNA extraction kit of New Industry company, the specific steps are as follows:
Using the bacterial genomes DNA extraction kit of New Industry company, the specific steps are as follows:
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 salmonella of New Ind μ stry company, extract
Genomic DNA carry out LAMP reaction immediately after preparation.In the heat inactivation salmonella sample of PMA processing, do not extract
Genomic DNA out, and sample cannot use LAMP to be expanded.And all may not be used using the life or death bacterium genomic DNA of PMA processing
To be expanded.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 it proves
PMA processing can effectively eliminate the DNA signal of dead bacterium.
4, design of primers
According to the conserved region for the salmonella gene invA that document is announced, primer is mediated by Japanese Rong Yan Co., Ltd. ring
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 2 LAMP of table
5, the amplification of salmonella gene group
(table 3) is expanded to salmonella gene group using ring mediated isothermal amplification method.
3 LAMP reaction system of table
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 salmonella gene group is not added as negative control group.5 μ L of amplified production is taken, with 1 μ L sample-loading buffer
It is uniformly mixed, carries out electrophoresis with 2% Ago-Gel, typical DNA cloning band (Fig. 1), nonsalmonella occurs in salmonella
Bacterial strain does not generate any band, thus proves that the primer pair salmonella 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 salmonella
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 salmonella 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 salmonella carries out mark-on reclaims verifying.Fresh chicken is bought from local supermarket, passes through standard culture after sterilizing
It is negative with colony counting method detection salmonella.Then by salmonella with 10,103With 105The concentration incorporation sterilizing of CFU/mL
It is mixed in chicken.PMA processing, genome extraction, LAMP amplification, nucleic acid test strip colour developing are successively carried out, peak area reads, is quantitative
Analysis, the rate of recovery of salmonella shows the present invention in the range of 101.3 ± 6.3% to 106.4 ± 8.2% as the result is shown
The sensor detecting method of foundation can be used for the detection (table 4) of actual sample.
4 nanometers of enzyme sensors of table detect chicken salmonella 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 invA 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.Salmonella living is being not present
In the case where bacterium, 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 salmonella (Fig. 3 A) the test biosensor of 100CFU/mL, survey altogether
Examination 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 always mixture of different bacterium type in practice, it is intended to make detection method that there is higher specificity.In order to evaluate
The specificity of nanometer enzyme sensor, using distilled water, the DNA of the dead bacterium of salmonella, the DNA of salmonella viable bacteria and other intestines bars
Campylobacter bacteria bacterial strain DNA and other non-Enterobacter bacteria bacterial strain DNA.As a result as shown in Figure 3B, false positive is not observed.Non- sand
The response holding of door Salmonella and the dead bacterium of salmonella is low as background signal, shows non-specific adsorption under the experiment condition
Salmonella living stems are had no significant effect to reaction system.Early period is by also further improving nanometer using LAMP amplification
The high specific of enzyme sensor.Stability (service life) is studied, is to test nanometer enzyme sensor after storing 1-5 weeks at room temperature
Performance.As a result as shown in Figure 3 C, nanometer enzyme sensor keeps almost the same to the reaction of 100CFU/mL salmonella, shows
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 Salmonella containing various concentration is measured under optimum experimental condition
Bacterium viable bacteria (range from 0 to 105CFU/mL sample solution) then measures the absorption peak area of TL line, and three parallel.In minimum
To maximum magnitude, the gained figure to the response of salmonella concentration be it is linear, and dependent equation be peak area=
25154lg salmonella concentration+4663, coefficient R2It is 0.9838, is suitble to quantitative detection.Wherein, salmonella concentration list
Position 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 salmonella
<130> KHP171113511.1
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
ggatgactcg ccatggtatg 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
ttgttcaaca gctgcgtca 19
<210> 3
<211> 39
<212> DNA
<213>artificial sequence
<400> 3
ctgggcgaca agaccatcac catttgtcct ccgccctgt 39
<210> 4
<211> 41
<212> DNA
<213>artificial sequence
<400> 4
tccccgcatt gttgattgcg atccgcccca tattatccgt a 41
Claims (9)
1. the LAMP primer group for detecting salmonella (Salmonella spp.) characterized by comprising
Outside forward primer F3:5 '-GGATGACTCGCCATGGTATG-3 '；
Outside reverse primer B3:5 '-TTGTTCAACAGCTGCGTCA-3 '；And
Inside forward primer FIP:5 '-CTGGGCGACAAGACCATCACCATTTGTCCTCCGCCCTGT-3 '；
Inside reverse primer BIP:5 '-TCCCCGCATTGTTGATTGCGATCCGCCCCATATTATCCGTA -3 '；
Wherein, 5 ' the end label biotins of primers F IP, 5 ' the end mark fluorescent element FITC of primer BIP.
2. the method based on nucleic acid chromatography biosensor technique detection salmonella for non-diagnostic purposes, which is characterized in that
The following steps are included:
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 salmonella 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；
The preparation method of the nanometer enzymatic nucleic acid chromatograph test strip the following steps are included:
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.
3. detecting the method for salmonella according to claim 2, which is characterized in that the nanometer enzymatic nucleic acid chromatograph test strip
Preparation method step 1) in using hydro-thermal method synthesize Fe3O4Magnetic particle, specifically: by 0.6-0.8g FeCl3·6H2O dissolution
In 20mL ethylene glycol, 1.5-2.0g sodium acetate is then added, stirs 30-40min, is then sealed in autoclave, 200 DEG C add
Hot 16-18h；Magnetic granular product ethanol washing, and it is dry at 60 DEG C；By dichloroethanes and each 5- of n-hydroxysuccinimide
8mg is dissolved in 1mL deionized water by being vortexed, and mixed liquor is made, and then 5-8mg magnetic particle is added in mixed liquor, room temperature
Then lower incubation 30-40min collects magnetic particle with magnet, with milli-Q water to get Fe3O4Magnetic particle.
4. detecting the method for salmonella according to claim 3, which is characterized in that the nanometer enzymatic nucleic acid chromatograph test strip
Preparation method step 2) specifically: the biotin secondary antibody of 100 μ g/mL of concentration is added to the sodium acetate buffer of 50mM pH 6.0
In liquid, then with the Fe of 5-8mg3O4Magnetic particle mixing, by mixture vortex mixed, 4 DEG C of overnight incubations；With the PBS liquid of pH7.0
Mixture is washed, is then incubated at room temperature 30-40min in the Tris buffer of 50mM pH7.2；It is washed again with the PBS liquid of pH7.0
It washs to get biotin secondary antibody nanometer enzyme probe is arrived.
5. detecting the method for salmonella according to claim 2, which is characterized in that the nanometer enzymatic nucleic acid chromatograph test strip
Preparation method step 2) and 3) described in biotin secondary antibody be sheep anti-mouse igg；Nitrocellulose filter described in step 3) is
Millipore135S。
6. according to the method for any one of the claim 2-5 detection salmonella, which is characterized in that the nanometer enzymatic nucleic acid layer
It analyses detection line in the preparation method step 3) of test strips to be located on the position away from nitrocellulose filter lower edge 1.1cm, nature controlling line
It is located on the position away from nitrocellulose filter lower edge 1.6cm；The distance between detection line and nature controlling line are 4.5mm, by 1.0 μ
FITC antibody and biotin antibody are sprayed in the detection line and nature controlling line of nitrocellulose filter by L/cm respectively, wherein detection line
It is 0.5-2mg/mL with antibody concentration coated on nature controlling line.
7. according to the method for any one of the claim 2-5 detection salmonella, which is characterized in that LAMP-PCR in step S1
The system of amplified reaction are as follows: 1 × Bst Thermal Buffer, 0.6M Betaine, 0.5mM dNTPs solution, 1.6 μM
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.
8. the method according to the description of claim 7 is characterized in that further including being folded to sample to be tested before step S1
The step of nitrogen propidium iodide processing, specifically: it is added in the sample to be tested for containing viable bacteria or hot inactivation of bacterial to 1 μ L final concentration of
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
Pipe is remotely from light source 20cm and is horizontally arranged on ice, shakes sample cell every 30s to keep to the uniform sudden and violent of light source
Dew；Then DNA extraction is carried out.
9. the kit containing LAMP primer group described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710481882.1A CN107340389B (en) | 2017-06-22 | 2017-06-22 | Method based on nucleic acid chromatography biosensor technique detection salmonella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710481882.1A CN107340389B (en) | 2017-06-22 | 2017-06-22 | Method based on nucleic acid chromatography biosensor technique detection salmonella |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107340389A CN107340389A (en) | 2017-11-10 |
| CN107340389B true CN107340389B (en) | 2019-01-29 |
Family
ID=60220047
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710481882.1A Active CN107340389B (en) | 2017-06-22 | 2017-06-22 | Method based on nucleic acid chromatography biosensor technique detection salmonella |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107340389B (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107365836A (en) * | 2017-06-22 | 2017-11-21 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Bacillus cereus |
| CN107385019A (en) * | 2017-06-22 | 2017-11-24 | 中国农业大学 | Method based on biosensor technique detection Listeria Monocytogenes |
| CN107340390B (en) * | 2017-06-22 | 2019-02-01 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection C.perfringens |
| CN107345961B (en) * | 2017-06-22 | 2019-01-29 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii |
| TR201820388A2 (en) | 2018-12-25 | 2019-01-21 | Tuerkiye Bilimsel Ve Teknolojik Arastirma Kurumu Tuebitak | FAST AND PORTABLE MICRO-FLUID DETECTION SYSTEM ALTERNATIVE TO THE CLASSIC CULTURAL METHOD OF SALMONELLAN |
| CN111007251B (en) * | 2019-12-23 | 2023-11-14 | 中国检验检疫科学研究院 | Method for detecting norovirus by nano enzyme test strip |
| CN112522379B (en) * | 2021-02-08 | 2021-05-11 | 中国农业大学 | Functional nucleic acid magnetic biosensor |
| CN113687071A (en) * | 2021-09-10 | 2021-11-23 | 佛山墨赛生物技术有限公司 | Salmonella typhimurium viable bacteria quantitative detection test strip, kit and detection method |
| CN114891903A (en) * | 2022-04-25 | 2022-08-12 | 华南农业大学 | Kit and method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103740803A (en) * | 2013-08-19 | 2014-04-23 | 天津出入境检验检疫局动植物与食品检测中心 | Food borne pathogenic organism nucleic acid lateral flow test strip detection kit and application thereof |
| 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 |
| CN107365836A (en) * | 2017-06-22 | 2017-11-21 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Bacillus cereus |
| CN107338291A (en) * | 2017-06-22 | 2017-11-10 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Escherichia coli O 157 |
| CN107385019A (en) * | 2017-06-22 | 2017-11-24 | 中国农业大学 | Method based on biosensor technique detection Listeria Monocytogenes |
| CN107340390B (en) * | 2017-06-22 | 2019-02-01 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection C.perfringens |
| CN107345961B (en) * | 2017-06-22 | 2019-01-29 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii |
-
2017
- 2017-06-22 CN CN201710481882.1A patent/CN107340389B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107340389A (en) | 2017-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107340389B (en) | Method based on nucleic acid chromatography biosensor technique detection salmonella | |
| CN107345961B (en) | Method based on nucleic acid chromatography biosensor technique detection Enterobacter sakazakii | |
| CN107340390B (en) | Method based on nucleic acid chromatography biosensor technique detection C.perfringens | |
| CN107419007A (en) | Method based on nucleic acid chromatography biosensor technique detection staphylococcus aureus | |
| CN107385019A (en) | Method based on biosensor technique detection Listeria Monocytogenes | |
| CN107338291A (en) | Method based on nucleic acid chromatography biosensor technique detection Escherichia coli O 157 | |
| CN110551846B (en) | Cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
| CN107365836A (en) | Method based on nucleic acid chromatography biosensor technique detection Bacillus cereus | |
| CN102181556B (en) | Kit for detecting equine piroplasmosis and preparation method and using method thereof | |
| CN110283936A (en) | A kind of African swine fever virus LAMP-HNB Visual retrieval kit | |
| CN110283935A (en) | A kind of African swine fever virus LAMP detection kit and its application | |
| CN107164458A (en) | A kind of multiple detection method of the malicious pseudomonas aeruginosa of field screening production | |
| Zeng et al. | A polymerase chain reaction based lateral flow test strip with propidium monoazide for detection of viable Vibrio parahaemolyticus in codfish | |
| US20220098645A1 (en) | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method | |
| CN108315450A (en) | The quickly primer of the close chlamydiosis of detection miscarriage and the kit constituted | |
| CN108977555B (en) | A kind of specific gene, detection method and the immuno-chromatographic test paper strip of fowl enteropathogenic E. Coli O78 serotype | |
| CN104263839A (en) | LAMP-LFD (loop-mediated isothermal amplification and lateral flow dipstick) detection kit and detection method for brucella | |
| CN106191286A (en) | Brucellar detection method, test kit and application thereof | |
| CN106544436B (en) | A kind of method of salmonella in detection textile | |
| Jiang et al. | Using dCas9 as an intermediate bridge of loop-mediated isothermal amplification-based lateral flow colorimetric biosensor for point-of-care Salmonella detection | |
| Edberg et al. | Measurement of active constitutive beta-D-glucosidase (esculinase) in the presence of sodium desoxycholate | |
| CN107271441A (en) | A kind of method of utilization dark field microscope direct visual perception Cryptosporidium | |
| US20190127811A1 (en) | Reagent and method for rapid detection of porcine adenovirus | |
| Huo et al. | A novel lateral flow assay for rapid and sensitive nucleic acid detection of Avibacterium paragallinarum | |
| CN108300795A (en) | The quickly primer of detection dog Ehlichiosis and detection reagent and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |