CN109298180A - A method of detection salmonella typhimurium - Google Patents
A method of detection salmonella typhimurium Download PDFInfo
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- CN109298180A CN109298180A CN201811377217.9A CN201811377217A CN109298180A CN 109298180 A CN109298180 A CN 109298180A CN 201811377217 A CN201811377217 A CN 201811377217A CN 109298180 A CN109298180 A CN 109298180A
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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Abstract
The invention discloses a kind of methods for detecting salmonella typhimurium, include the steps that following: making carboxymethyl chitosan in conjunction with the specific aptamers sequence of salmonella typhimurium, carboxymethyl chitosan-adaptor complex is formed, described specificity aptamers sequence one end is with amido modified；Carboxymethyl chitosan-adaptor complex is added in gold nano solution, compound is adsorbed on gold nano surface, obtains carboxymethyl chitosan-aptamers-gold nano-material；Sample to be tested is mixed with carboxymethyl chitosan-aptamers-gold nano-material, qualitative detection or use ultraviolet-visible spectrophotometry quantitative detection salmonella typhimurium.The raw materials used in the present invention is at low cost, from a wealth of sources, and improves into born of the same parents' efficiency, without extracting the DNA of salmonella typhimurium, directly bacterium itself can be detected, shorten detection time, it is a kind of quick, economic, the convenient and fast method for detecting salmonella typhimurium.
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of specific biological sensor for detecting mouse typhus sramana
The method of Salmonella.
Background technique
Salmonella typhimurium is a kind of Grain-negative anaerobic bacteria, is widely present in natural environment, can be by contaminated
Poultry, egg, milk, fish and meat products infect the mankind, may colonize in people after the almost all of serotype infection mankind
In body or the enteron aisle of animal.Traditional detection method is food samples distribution enrichment, including increases bacterium in advance, selective enrichment, point
From culture and biochemical serological Identification.The result that this method obtains is accurate, but takes a long time, and generally requires 4-7 talent
It can be obtained definite result, other methods such as rely on the ELISA method of antigen-antibody reaction, vulnerable to pH, the environment shadow such as temperature when reaction
It rings, and antigen-antibody synthesis is expensive.
Carboxymethyl chitosan is the derivative of chitosan, it is modified it on the basis of chitosan, and carboxymethyl exists
Replace on hydroxyl or amino, by changing preparation condition, O- carboxymethyl can be formed according to the different location of substituent group
Chitosan, N- carboxymethyl chitosan or O, N- carboxymethyl chitosan.Carboxymethyl chitosan is a kind of carboxylic acid sodium salt, thus is had
Water solubility, and carboxymethyl is introduced again, the structure of chitosan itself is changed, so that its water solubility be made to reinforce.Carboxymethyl chitosan
Sugar has stable property, and antibacterial anti-infection is strong, and for many years in chemical industry, medicine, there are many applications for environmental protection etc..
Gold nano is that partial size is generally 15nm microparticle below.Gold nano stable structure, particle size is small, can be with many
Large biological molecule combines, and will not change its bioactivity after combining, and can be used for drug therapy in human body, is a kind of extensive use
In the substance with good optical, electrochemical properties of every field.
Aptamers are the oligonucleotides sequences with high specific and high-affinity to target gone out by SELEX technology screening
Column.Aptamers have the advantages that various, are such as readily synthesized, and stability is high, reproducible, cheap, and aptamers target
Extensively, with cell, virus, toxin, protein and vitamin etc. can be specifically bound.Due to these advantages of aptamers, closely
Nian Lai, aptamers will be used wider and wider.In context of detection, since aptamers can be with various bacteria, toxin equiconjugate shape
At compound, to realize the detection to it, this is a kind of quickly and easily detection means, and have good sensitivity and
Selectivity, compared with traditional detection method, detection cycle is short, and process is simple.
Since aptamers have specificity to object bacteria, targeting detection can be carried out, gold nano generates color under salting liquid
The characteristic of variation, adaptation body technique combination gold nano carries out Visual retrieval and is possibly realized, but aptamers itself are oligonucleotides,
It is difficult to the cell membrane across surface negative charge, enters born of the same parents and leads very low, existing method is to extract the DNA of salmonella typhimurium, then right
It is detected.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the shortcomings of to mention in background above technology and defect, provide one
Kind is quick, sensitive, low in cost, and can solve the method that too low Visual retrieval salmonella typhimurium is led into born of the same parents.
In order to solve the above technical problems, technical solution proposed by the present invention are as follows:
A method of detection salmonella typhimurium includes the steps that following:
Make carboxymethyl chitosan in conjunction with the specific aptamers sequence of salmonella typhimurium, forms carboxymethyl chitosan
Sugar-adaptor complex, described specificity aptamers sequence one end is with amido modified；
Carboxymethyl chitosan-adaptor complex is added in gold nano solution, compound is adsorbed on gold nano surface,
Obtain carboxymethyl chitosan-aptamers-gold nano-material；
Sample to be tested is mixed with carboxymethyl chitosan-aptamers-gold nano-material, qualitative detection or use UV, visible light
Spectrophotometry quantitative detection salmonella typhimurium.
Further, the carboxymethyl chitosan is O, N- carboxymethyl chitosan.
Further, the specific aptamers sequence is crosslinking cyclic structure.Specific aptamers nucleotides sequence is classified as
NH2-GCGCTCGGCCTCCTCTGCCATCTCATTCGCGAGCGC。
Further, carboxymethyl chitosan and the mass ratio of aptamers are 3~5:1.
Further, before carboxymethyl chitosan is in conjunction with specific aptamers sequence, first carboxymethyl chitosan is carried out
Following processing make activated carboxylic: n-hydroxysuccinimide is added and carries out first step reaction, is then added with stirring 1- ethyl -3-
(dimethylaminopropyl) carbodiimide carries out second step reaction.Second step reaction carries out under ice-water bath.
Further, carboxymethyl chitosan, n-hydroxysuccinimide and 1- ethyl -3- (dimethylaminopropyl) carbon two
Imines, the mass ratio of three are 0.28~0.32:0.15~0.18:0.24~0.28.
Further, carboxymethyl chitosan-adaptor complex is incubated for 20~35h after gold nano solution is added.
Further, 20~30min is incubated for after sample to be tested is mixed with carboxymethyl chitosan-aptamers-gold nano-material,
Then inorganic salts, which are added, makes gold nano grain reunite.
The present invention is to be based on following technical principle, as shown in Figure 1: the aptamers sequence of specific salmonella typhimurium
Can specific recognition salmonella typhimurium and with its stable bond.And gold nano grain can pass through electrostatic interaction and aptamers knot
It closes, forms the targeting compound of energy specific detection salmonella typhimurium.
The reason of by carboxymethyl chitosan in conjunction with the compound is: only aptamers are formed compound with gold nano grain
The surface of object is negative electrical charge, it is difficult to across the cell membrane of surface negative charge, easily be degraded by nuclease, so itself not can enter thin
Born of the same parents need ideal transport vehicle to be carried along into cell.Therefore it uses carboxymethyl chitosan-adaptor complex of positive charge and bears
The gold nano of charge is solved the problems, such as into born of the same parents' low efficiency.There are a large amount of carboxyls and amino, carboxyl on the carboxymethyl chitosan of selection
It is combined with amido modified aptamers, aptamers are combined by electrostatic interaction and gold nano again, and entire material is because of carboxymethyl chitosan
On amino and it is positively charged, material can be improved enters born of the same parents' efficiency, realizes and does not have to extract DNA, can directly detect to bacterium solution.
Compared with prior art, advantages of the present invention:
1. the present invention is for the first time carrier using carboxymethyl chitosan, in conjunction with salmonella typhimurium specificity aptamers, with
Gold nano solution is visualization index, and biosensor type detection architecture collection high efficiency, the detection time of design are short, at low cost etc.
Plurality of advantages, while having high specific and the visual advantage of testing result, can pass through the direct qualitative observation of naked eyes
Testing result can exclusively detect salmonella typhimurium, and reduce sample pre-treatments process, can be directly right
Bacterium solution is detected.
2. the present invention carries aptamers-gold nano using its surface charge as transport vehicle using carboxymethyl chitosan
Compound enters cell, solves the problems, such as into born of the same parents' low efficiency, does not have to extract DNA to reach, can be directly to Salmonella typhimurium
The bacterium solution of bacterium is detected.
3. the changed property of the gold nano solution that the present invention uses color in high level salt solution, has testing result
It is visual.The specific aptamers of selection, source is wide, at low cost, largely improves the economic benefit of the method, can be special
Property identification salmonella typhimurium, can to salmonella typhimurium carry out quantitative detection.
To sum up, the raw materials used in the present invention is at low cost, from a wealth of sources, and improves into born of the same parents' efficiency, does not have to extract mouse typhus
The DNA of salmonella can directly detect bacterium itself, shorten detection time, be a kind of quick, economy, easily
The method for detecting salmonella typhimurium, can be widely used for the detection of salmonella typhimurium in food.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the reaction principle figure of carboxymethyl chitosan of the invention, aptamers and gold nano；
Fig. 2 be in embodiment 2 under different incubation times detection effect of the reaction system to salmonella typhimurium.
Specific embodiment
To facilitate the understanding of the present invention, the present invention is done below in conjunction with Figure of description and preferred embodiment more complete
Face meticulously describes, but protection scope of the present invention is not limited to following specific embodiments.
Unless otherwise defined, all technical terms used hereinafter are generally understood meaning phase with those skilled in the art
Together.Technical term used herein is intended merely to the purpose of description specific embodiment, and it is of the invention to be not intended to limitation
Protection scope.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention can pass through city
Field is commercially available or can be prepared by existing method.
Fig. 1 is the reaction principle figure of carboxymethyl chitosan of the invention, aptamers and gold nano.The wound of mouse used in the present invention
The specific aptamers sequence of cold salmonella, is commercially available.In one end with amido modified, so as to the carboxylic with chitosan
Base reaction bonded.It is preferably crosslinked cyclic structure, aptamers are more stable.Specificity aptamers nucleotides sequence used in following embodiments
It is classified as NH2-GCGCTCGGCCTCCTCTGCCATCTCATTCGCGAGCGC。
Carboxymethyl chitosan used in the present invention is preferably O, N- carboxymethyl chitosan.With O-CMC and N- carboxylic first
Base enclosure glycan is compared, O, and the carboxymethyl of N- carboxymethyl chitosan replaces on-OH and-NH, can introduce more carboxylics
Methyl improves carboxymethyl chitosan convenient for preferably being combined with amido modified aptamers, and due to the introducing of carboxymethyl
It is water-soluble.
Before carboxymethyl chitosan is in conjunction with specific aptamers sequence, carboxymethyl chitosan, which is first carried out following processing, makes
Activated carboxylic, the mechanism are as follows:-NH in EDC2Acid-base neutralization reaction occurs with carboxymethyl, the hydroxyl in carboxymethyl lose hydrogen from
Son, becomes the state of activation, and NHS is usually combined with EDC, can be used for improving reaction efficiency.
Embodiment 1:
The method of the detection salmonella typhimurium of the present embodiment, specifically includes the following steps:
(1) it prepares gold nano solution: drawing 1% gold chloride 1.65ml in 250ml conical flask, 38.35ml water is added, it will
Conical flask, which is placed in magnetic heating stirrer, be protected from light reflux (connecing condenser pipe on conical flask), is put into stirrer in conical flask,
Magnetic agitation simultaneously is boiled to solution, and the citric acid three sodium solution of 6.4ml 38.8mmol/L is added immediately, continues to be protected from light back
Stream, heating stirring keep boiling 15min.Heat source is removed, is stirred to cooling, the nanogold for obtaining the dispersity of claret is molten
Liquid.After filtering gold nano solution with 0.22 μm of needle-based water solubility filter, it is placed in 125mL brown ground reagent bottle, aluminium foil
Paper bag is wrapped up in, and is protected from light 4 DEG C and is saved backup.
(2) it prepares carboxymethyl chitosan-adaptor complex: taking carboxymethyl chitosan (purchased from BELLANCOM LIFE
SCIENCES DEP, 100g/ bottle) 0.3g is in 50ml beaker, and addition 10ml deionized water, it is dense that glass bar stirring and dissolving obtains quality
Degree is the carboxymethyl chitosan solution of 30mg/ml.N-hydroxysuccinimide (NHS) 0.17g, stirring are added into above-mentioned solution
15min is stirred down then in 2 DEG C of ice-water bath blenders with 800r/min, is slowly dropped into 1- ethyl -3- (dimethylamino third
Base) carbodiimide (EDC) aqueous solution (0.26g EDC in 15ml centrifuge tube, be added 5ml deionized water dissolving).10min drop
After the completion of adding, still continues to be stirred to react 1h under ice-water bath, activate the carboxyl of carboxymethyl chitosan sufficiently.By carboxymethyl chitosan
Sugar and the mass ratioes of aptamers are 4:1, be added salmonella typhimurium specific aptamers (purchased from raw work bioengineering (on
Sea) limited liability company, 5 ' ends have amido modified) and into above-mentioned solution, handle 1h.It is multiple to obtain carboxymethyl chitosan-aptamers
Close object.
(3) it prepares carboxymethyl chitosan-aptamers-gold nano-material: 1mL self-control nano-Au solution being taken to manage in 1.5mL EP
In, 4 DEG C, 10000r/min centrifugation 10min abandon supernatant, are resuspended.The carboxymethyl chitosan prepared is added into above-mentioned nano-Au solution
100 μ L of sugar-adaptor complex is mixed, is placed in 37 DEG C of shaking tables and is incubated for 30h.It, will be above-mentioned to remove possible excessive aptamers
Mixed liquor is centrifuged 10min in 4 DEG C, 10000r/min, abandons supernatant, finally obtains carboxymethyl chitosan-aptamers-nanogold material
Material, 4 DEG C be kept in dark place it is spare.
Salmonella typhimurium sample detection:
It takes the EP of 9 1.5ml to manage, the 100 μ L of carboxymethyl chitosan-molecular beacon-golden nano-complexes prepared is added, adds
Enter salmonella typhimurium bacterium solution, 6 dilutions of doubling dilution are respectively 1:10,1:102, 1:103, 1:104, 1:105With 1:
106, (bacterial concentration 10cfu/ml, 102cfu/ml、103cfu/ml、104cfu/ml、105cfu/ml、106Cfu/ml) in 37
DEG C be incubated for 30min.
The NaCl solution that 1.5mol/L is added makes the gold nano grain released reunite, and is examined with ultraviolet-uisible spectrophotometer
The absorbance A value of maximum absorption wave strong point is surveyed, standard curve is drawn, is quantitative determined.
Embodiment 2:
The method of the detection salmonella typhimurium of the present embodiment, specifically includes the following steps:
(1) it prepares gold nano solution: drawing 1% gold chloride 1.65ml in 250ml conical flask, 38.35ml water is added, it will
Conical flask, which is placed in magnetic heating stirrer, be protected from light reflux (connecing condenser pipe on conical flask), is put into stirrer in conical flask,
Magnetic agitation simultaneously is boiled to solution, and the citric acid three sodium solution of 6.4ml 38.8mmol/L is added immediately, continues to be protected from light back
Stream, heating stirring keep boiling 15min.Heat source is removed, is stirred to cooling, the nanogold for obtaining the dispersity of claret is molten
Liquid.After filtering gold nano solution with 0.22 μm of needle-based water solubility filter, it is placed in 125mL brown ground reagent bottle, aluminium foil
Paper bag is wrapped up in, and is protected from light 4 DEG C and is saved backup.
(2) it prepares carboxymethyl chitosan-adaptor complex: taking carboxymethyl chitosan (purchased from BELLANCOM LIFE
SCIENCES DEP, 100g/ bottle) 0.3g is in 50ml beaker, and addition 10ml deionized water, it is dense that glass bar stirring and dissolving obtains quality
Degree is the carboxymethyl chitosan solution of 30mg/ml.N-hydroxysuccinimide (NHS) 0.17g, stirring are added into above-mentioned solution
15min is stirred down then in 2 DEG C of ice-water bath blenders with 800r/min, is slowly dropped into 1- ethyl -3- (dimethylamino third
Base) carbodiimide (EDC) aqueous solution (0.26g EDC in 15ml centrifuge tube, be added 5ml deionized water dissolving).10min drop
After the completion of adding, still continues to be stirred to react 1h under ice-water bath, activate the carboxyl of carboxymethyl chitosan sufficiently.By carboxymethyl chitosan
Sugar and the mass ratioes of aptamers are 4:1, be added salmonella typhimurium specific aptamers (purchased from raw work bioengineering (on
Sea) limited liability company, 5 ' ends have amido modified) and into above-mentioned solution, handle 1h.It is multiple to obtain carboxymethyl chitosan-aptamers
Close object.
(3) it prepares carboxymethyl chitosan-aptamers-gold nano-material: 1mL self-control nano-Au solution being taken to manage in 1.5mL EP
In, 4 DEG C, 10000r/min centrifugation 10min abandon supernatant, are resuspended.The carboxymethyl chitosan prepared is added into above-mentioned nano-Au solution
100 μ L of sugar-adaptor complex, mix, be placed in 37 DEG C of shaking tables be incubated for respectively 12h, 18h, for 24 hours, 30h, 36h and 42h.For except
Possible excessive aptamers are gone, above-mentioned mixed liquor is centrifuged 10min in 4 DEG C, 10000r/min, supernatant is abandoned, finally obtains carboxylic first
Base enclosure glycan-aptamers-nanogold material, 4 DEG C be kept in dark place it is spare.
Salmonella typhimurium sample detection:
It takes the EP of 9 1.5ml to manage, the 100 μ L of carboxymethyl chitosan-molecular beacon-golden nano-complexes prepared is added, adds
Enter salmonella typhimurium bacterium solution, 6 dilutions of doubling dilution are respectively 1:10,1:102, 1:103, 1:104, 1:105With 1:
106, in 37 DEG C of incubation 30min.
The NaCl solution that 1.5mol/L is added makes the gold nano grain released reunite, and is examined with ultraviolet-uisible spectrophotometer
The absorbance A value of maximum absorption wave strong point is surveyed, standard curve is drawn, is quantitative determined.
The reaction system that the present embodiment obtains is to the detection effect of salmonella typhimurium DNA sequence dna as shown in Fig. 2, by scheming
It is found that detection effect is best when incubation time is 30h.
Embodiment 3:
The method of the detection salmonella typhimurium of the present embodiment, specifically includes the following steps:
(1) it prepares gold nano solution: drawing 1% gold chloride 1.65ml in 250ml conical flask, 38.35ml water is added, it will
Conical flask, which is placed in magnetic heating stirrer, be protected from light reflux (connecing condenser pipe on conical flask), is put into stirrer in conical flask,
Magnetic agitation simultaneously is boiled to solution, and the citric acid three sodium solution of 6.4ml 38.8mmol/L is added immediately, continues to be protected from light back
Stream, heating stirring keep boiling 15min.Heat source is removed, is stirred to cooling, the nanogold for obtaining the dispersity of claret is molten
Liquid.After filtering gold nano solution with 0.22 μm of needle-based water solubility filter, it is placed in 125mL brown ground reagent bottle, aluminium foil
Paper bag is wrapped up in, and is protected from light 4 DEG C and is saved backup.
(2) it prepares carboxymethyl chitosan-adaptor complex: taking carboxymethyl chitosan (purchased from BELLANCOM LIFE
SCIENCES DEP, 100g/ bottle) 0.3g is in 50ml beaker, and addition 10ml deionized water, it is dense that glass bar stirring and dissolving obtains quality
Degree is the carboxymethyl chitosan solution of 30mg/ml.N-hydroxysuccinimide (NHS) 0.17g, stirring are added into above-mentioned solution
15min is stirred down then in 2 DEG C of ice-water bath blenders with 800r/min, is slowly dropped into 1- ethyl -3- (dimethylamino third
Base) carbodiimide (EDC) aqueous solution (0.26g EDC in 15ml centrifuge tube, be added 5ml deionized water dissolving).10min drop
After the completion of adding, still continues to be stirred to react 1h under ice-water bath, activate the carboxyl of carboxymethyl chitosan sufficiently.By carboxymethyl chitosan
Sugar and the mass ratioes of aptamers are 4:1, be added salmonella typhimurium specific aptamers (purchased from raw work bioengineering (on
Sea) limited liability company, 5 ' ends have amido modified) and into above-mentioned solution, handle 1h.It is multiple to obtain carboxymethyl chitosan-aptamers
Close object.
(3) it prepares carboxymethyl chitosan-aptamers-gold nano-material: 1mL self-control nano-Au solution being taken to manage in 1.5mL EP
In, 4 DEG C, 10000r/min centrifugation 10min abandon supernatant, are resuspended.The carboxymethyl chitosan prepared is added into above-mentioned nano-Au solution
100 μ L of sugar-adaptor complex is mixed, is placed in 37 DEG C of shaking tables and is incubated for 30h.It, will be above-mentioned to remove possible excessive aptamers
Mixed liquor is centrifuged 10min in 4 DEG C, 10000r/min, abandons supernatant, finally obtains carboxymethyl chitosan-aptamers-nanogold material
Material, 4 DEG C be kept in dark place it is spare.
Salmonella typhimurium sample detection:
It takes the EP of 9 1.5ml to manage, the 100 μ L of carboxymethyl chitosan-molecular beacon-golden nano-complexes prepared is added, adds
Enter salmonella typhimurium bacterium solution, 6 dilutions of doubling dilution are respectively 1:10,1:102, 1:103, 1:104, 1:105With 1:
106, in 37 DEG C of incubation 30min.
The NaCl solution that 1.5mol/L is added makes the gold nano grain released reunite, and is examined with ultraviolet-uisible spectrophotometer
The absorbance A value of maximum absorption wave strong point is surveyed, standard curve is drawn, is quantitative determined.
Above-mentioned only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form.Therefore, it is all not
Be detached from technical solution of the present invention content, according to the present invention technical spirit it is made to the above embodiment it is any it is simple modification, etc.
With variation and modification, all shall fall within the protection scope of the technical scheme of the invention.
Claims (10)
1. a kind of method for detecting salmonella typhimurium, which is characterized in that include the steps that following:
Make carboxymethyl chitosan in conjunction with the specific aptamers sequence of salmonella typhimurium, it is suitable to form carboxymethyl chitosan-
Ligand complex, described specificity aptamers sequence one end is with amido modified；
Carboxymethyl chitosan-adaptor complex is added in gold nano solution, compound is adsorbed on gold nano surface, obtains
Carboxymethyl chitosan-aptamers-gold nano-material；
Sample to be tested is mixed with carboxymethyl chitosan-aptamers-gold nano-material, qualitative detection or use UV, visible light are divided
Photometry quantitative detection salmonella typhimurium.
2. the method for detection salmonella typhimurium according to claim 1, which is characterized in that the carboxymethyl chitosan
For O, N- carboxymethyl chitosan.
3. the method for detection salmonella typhimurium according to claim 1, which is characterized in that the specificity aptamers
Sequence is crosslinking cyclic structure.
4. detecting the method for salmonella typhimurium described according to claim 1~one of 3, which is characterized in that specificity is suitable
Ligand nucleotides sequence is classified as NH2-GCGCTCGGCCTCCTCTGCCATCTCATTCGCGAGCGC。
5. the method for detection salmonella typhimurium according to claim 1, which is characterized in that carboxymethyl chitosan and suitable
The mass ratio of ligand is 3~5:1.
6. according to claim 1, detecting the method for salmonella typhimurium described in 2 or 5, which is characterized in that in carboxymethyl shell
Before glycan is in conjunction with specific aptamers sequence, carboxymethyl chitosan, which is first carried out following processing, makes activated carboxylic: N- hydroxyl is added
Base succinimide carries out first step reaction, is then added with stirring 1- ethyl -3- (dimethylaminopropyl) carbodiimide, into
The reaction of row second step.
7. the method for detection salmonella typhimurium according to claim 6, which is characterized in that second step is reacted in ice water
Bath is lower to be carried out.
8. the method for detection salmonella typhimurium according to claim 6, which is characterized in that carboxymethyl chitosan, N-
The mass ratio of HOSu NHS and 1- ethyl -3- (dimethylaminopropyl) carbodiimide, three is 0.28~0.32:
0.15~0.18:0.24~0.28.
9. detecting the method for salmonella typhimurium described according to claim 1~one of 3, which is characterized in that carboxymethyl shell
Glycan-adaptor complex is incubated for 20~35h after gold nano solution is added.
10. detecting the method for salmonella typhimurium described according to claim 1~one of 3, which is characterized in that sample to be tested
20~30min is incubated for after mixing with carboxymethyl chitosan-aptamers-gold nano-material, inorganic salts, which are then added, makes gold nano
Grain is reunited.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112263490A (en) * | 2020-11-24 | 2021-01-26 | 上海健康医学院 | Toothpaste for developing oral helicobacter pylori and preparation method thereof |
| CN115856297A (en) * | 2023-01-04 | 2023-03-28 | 吉林大学 | Preparation method of kit for detecting salmonella typhimurium and kit |
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