CN106053805A - Sensitivity enhanced type nucleic acid aptamer test strip for detecting salmonella and method for preparing sensitivity enhanced type nucleic acid aptamer test strip - Google Patents
Sensitivity enhanced type nucleic acid aptamer test strip for detecting salmonella and method for preparing sensitivity enhanced type nucleic acid aptamer test strip Download PDFInfo
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- CN106053805A CN106053805A CN201610582015.2A CN201610582015A CN106053805A CN 106053805 A CN106053805 A CN 106053805A CN 201610582015 A CN201610582015 A CN 201610582015A CN 106053805 A CN106053805 A CN 106053805A
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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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- 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
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- 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 sensitivity enhanced type nucleic acid aptamer test strip for detecting salmonella and a method for preparing the sensitivity enhanced type nucleic acid aptamer test strip, and belongs to the field of food safety detection. The sensitivity enhanced type nucleic acid aptamer test strip comprises a sample pad, a gold standard pad, an envelope membrane and a water absorption membrane. The sample pad, the gold standard pad, the envelope membrane and the water absorption membrane are sequentially adhered on a PVC (polyvinyl chloride) bottom plate in a combined manner, dendronized polymer-gold-aptamer compound sensitivity enhanced probes are coated on the gold standard pad, a detection line and a quality control line are arranged on the envelop membrane, salmonella-resistant monoclonal antibodies are enveloped on the detection line, and streptavidin is enveloped on the quality control line. The method for preparing the sensitivity enhanced type nucleic acid aptamer test strip includes preparing the dendronized polymer-gold-aptamer compound sensitivity enhanced probes; preparing the gold standard pad; preparing the envelop membrane; assembling the sensitivity enhanced type nucleic acid aptamer test strip and the like. The sensitivity enhanced type nucleic acid aptamer test strip and the method have the advantages that signals can be amplified by the aid of the dendronized polymer-gold-aptamer compound sensitivity enhanced probes, and accordingly the detection sensitivity can be greatly improved; the sensitivity enhanced type nucleic acid aptamer test strip is easy to operate, high in detection speed, low in cost and suitable for quickly detecting salmonella in a large amount of samples in a high-sensitivity manner, and special instruments and equipment can be omitted.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to a kind of sensitizing type nucleic acid for detecting Salmonella and fit
Part test strips and preparation method thereof.
Background technology
Salmonella belongs to gram negative bacteria, is a kind of Bacterium entericum pathogenic bacterium, is widespread in nature, easily
Polluted source, meat (especially birds), eggs and egg products etc., human or animal eats contaminated food i.e. can cause food
Poisoning.At present, in worldwide, particularly developing country's Salmonella infection event increases year by year, China 70～
The bacterial food poisoning of 80% is by salmonellal, it is seen that salmonella-polluted serious threat is to human health
With public food safe and sanitary.Therefore, Salmeterol fluticasone propionate technology accurate, sensitive, quick is set up for ensuring food safety
Significant with national health.
At present Detection Methods of Salmonella mainly has traditional microbial testing technology, instrumental method, molecular biology
Technology and immunologic detection method etc..Wherein, there is operation complexity in traditional microorganism detection technology, the detection time is long, sensitivity
With the shortcoming such as specificity is the highest；Instrumental method is simple and quick, but instrument and equipment is expensive, is not suitable for field quick detection；
Molecular biology method can shorten Check-Out Time, and sensitivity and specificity are good, but somewhat expensive, easily to pollute, false positive is higher；
ELISA method is sensitive, quick, specificity is good, has been widely used in the detection of microorganism in food, but its result is understood or
Necessary instrument is more complicated, needs certain Professional knowledge.
And gold mark chromatograph test strip just can avoid these drawbacks, it is not necessary to instrumentation, it is only necessary to insert, comparison is
Can, there is quick, low cost, operation is simple, can meet the detection of overwhelming majority food samples, be highly suitable for a large amount of sample
The primary dcreening operation of product.But, conventional gold label test strip sensitivity is relatively low, it is difficult to realize the quick analysis of trace object.Meanwhile, with
Improving and the reinforcement of inspecting force of governmental supervision, in food, the concentration of target detection thing can be fewer and feweri, and this is to gold
The sensitivity of mark test strips is had higher requirement.
Summary of the invention
The sensitivity that the present invention is directed to existing gold label test strip is difficult to meet the present Research of the highly sensitive detection of Salmonella,
Dendritic is utilized to amplify carrier as signal, by dendritic, golden nanometer particle, aptamer
(Aptamer) assemble, prepare dendritic-gold-aptamer nano-complex, visit as signal enhanced sensitivity
Pin is applied in gold label test strip, carries out gold label test strip detection signal and strengthens research, it is provided that one is used for detecting Salmonella
Sensitizing type aptamer test strips and preparation method thereof, it is achieved the high-sensitivity rapid detection to Salmonella in Food.
Technical scheme is as follows:
A kind of sensitizing type aptamer test strips for detecting Salmonella includes PVC base plate, at described PVC base plate
On be pasted with sample film, signal enhanced sensitivity probe-gold-marking binding pad, nitrocellulose filter and four kinds of paste of absorbing membrane successively；Institute
State to be connected with each other between adjacent paste and stack；Detection line and nature controlling line it is disposed with on described nitrocellulose filter.
It is coated with dendritic-gold particle-aptamer nanometer on described signal enhanced sensitivity probe-gold-marking binding pad
Complex；Described dendritic is the dendritic of end sulfydryl；The 5 ' of described aptamer are held by marking sulfhydryl,
3 ' ends are by biotin labeling.
It is coated with anti-salmonella monoclonal antibody on described detection line.
It is coated with Streptavidin on described nature controlling line.
Described for sensitizing type aptamer test strips detecting Salmonella and preparation method thereof, it specifically prepares step
Rapid as follows:
(1) preparation of dendritic-golden nanometer particle complex
The dendritic weighing 0.1-0.4mg is dissolved in 5mL acetone, and taking newly synthesized particle diameter is 10-20nm's
Colloidal gold solution 1mL colloidal gold solution is distributed in 5mL pure water, in nitrogen protection with quickly under stirring, by colloidal gold solution by
It is added drop-wise in dendrimer solution, under magnetic agitation after room temperature reaction 12h, 4 DEG C of centrifugal 30min of 10000rpm, abandon
Clearly, use pure water precipitate, centrifuge washing and after being repeated 3 times by precipitation re-suspension liquid (20mM Na3PO4, 5%BSA,
0.25%Tween-20,10% sucrose) resuspended, obtain 10 times of dendritic-golden nanometer particle complex solutions concentrated.
(2) preparation of dendritic-gold particle-aptamer complex
The aptamer taking 10uL 1uM joins 2uL acetate buffer solution (0.5M, pH5.0) and 3uL 1mM tricarboxylic first
In base phosphoric acid solution, room temperature lucifuge places 1h, obtains the aptamer solution of activation.
Take 10 times of dendritic-golden nanometer particle complex solution 200uL concentrated, be added dropwise to the core of activation
In acid aptamers solution, under room temperature, stir 60min；The dATP taking 170uL 100uM joins in above-mentioned reactant liquor, continues under room temperature
Continuous stirring 30min；Progressively the NaCl solution of 0.1M is joined in reactant liquor, make the final concentration of NaCl reach 30mM, under room temperature
Place after reaction 1h and hatch with 4 DEG C of refrigerator overnight；It is resuspended to 200uL that 9400g is centrifuged 10min postprecipitation re-suspension liquid, is set
Dendritic polymer-gold particle-aptamer complex.
Salmonella nucleic acid aptamer sequence is: 5 '-SH-TAT GGC GGC GTC ACC CGA CGG GGA CTT
GAC ATT ATG ACA G-biotin-3 ', is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
(3) preparation of dendritic-gold particle-aptamer enhanced sensitivity probe gold-marking binding pad
With three-dimensional planar point film gold spraying instrument by the dendritic-gold particle-aptamer complex of above-mentioned preparation
Uniformly spraying on glass fibre membrane, spouting liquid is 0.7uL/cm, is placed in 37 DEG C of drying post packages standby.
(4) preparation of coated film
With three-dimensional planar point film gold spraying instrument by uniform for the anti-salmonella monoclonal antibody solution that concentration is 0.5-1mg/mL
Spray on nitrocellulose filter, obtain detect line (T line)；By uniform for solution of streptavidin that concentration is 0.5-2mg/mL
Spray on nitrocellulose filter, obtain nature controlling line (C line).
(5) preparation of sensitizing type Salmonella aptamer chromatograph test strip
By sample film, dendritic-gold particle-aptamer enhanced sensitivity probe gold-marking binding pad, there is detection line
With on the nitrocellulose filter of nature controlling line and adsorptive pads paste composition successively to PVC base plate, the overlapping portion between adjacent paste
Divide a length of 2mm, be cut into the wide test strips of 3mm with cutting machine, obtain sensitizing type Salmonella aptamers based on enhanced sensitivity probe
Test strips, is loaded in aluminium foil bag sealing together with desiccant and saves backup.
Test strips of the present invention uses colloid gold chromatographic test paper technology, with dendritic as carrier, utilizes it peripheral
Substantial amounts of sulfydryl, can a large amount of golden nanometer particle of coupling by gold sulfide linkage self assembly.When testing sample exists Salmonella, husky
Door Salmonella dendritic-gold particle-aptamer complex specific bond first and on gold-marking binding pad, along with capillary
Pipe effect continues chromatography on nitrocellulose filter, then with the antibody generation Immune discrimination of coated anti-salmonella on detection line
Reaction, shows the precipitation line of certain color on detection line；Unnecessary dendritic-gold particle-aptamer is multiple
Compound continues forward, and on aptamer, on the biotin of labelling and nature controlling line, coated Streptavidin combines, Jenner
Rice corpuscles enrichment makes nature controlling line develop the color；If there is not Salmonella in testing sample, dendritic-gold particle-
Aptamer complex does not produce enrichment on detection line, and the Streptavidin continued on chromatography migration and nature controlling line is tied
Closing, ELISA test strip line does not develops the color, and nature controlling line colour developing.
The detection line of test strips and nature controlling line are all to be assembled by golden nanometer particle and develop the color, compared with conventional test strips, when
When Salmonella concentration is extremely low, detection line also can be enriched with more golden nanometer particle, the colour developing of its precipitation line also can be deeper, because of
This, can realize the super sensitivity detection than conventional test strips lower concentration object, significantly improve the sensitive of ELISA test strip method
Degree.
Beneficial effects of the present invention: the sensitivity that the present invention is directed to existing gold label test strip is difficult to meet Salmonella Gao Ling
The requirement of quick detection, it is provided that a kind of for sensitizing type aptamer test strips detecting Salmonella and preparation method thereof, borrows
Help the end sulfydryl of dendritic and carry out the substantial amounts of golden nanometer particle of coupling, further with the core of specific recognition Salmonella
Dendritic-gold-nucleic probe is prepared in acid aptamers assembling, and replaces conventional gold-nucleic acid as signal enhanced sensitivity probe
Aptamers probe, it is possible to significantly improve the sensitivity of tradition ELISA test strip method, it is achieved the highly sensitive detection to Salmonella,
There is the features such as simple, quick, the easy interpretation of result simultaneously.
Accompanying drawing explanation
The composition structural representation of Fig. 1 test strips of the present invention.
The Cleaning Principle schematic diagram of Fig. 2 test strips of the present invention.
The testing result process decision chart of Fig. 3 test strips of the present invention.
Sequence number explanation in accompanying drawing: 1:PVC base plate；2: sample pad；3: enhanced sensitivity probe gold-marking binding pad；4: celluloid
Film；5: adsorptive pads；6: detection line；7: nature controlling line.
Detailed description of the invention
For a better understanding of the present invention, it is further elucidated with below in conjunction with specific embodiment, it should be appreciated that these embodiments
It is merely to illustrate the present invention, and is not limited to the scope of the present invention.
Embodiment 1
As it is shown in figure 1, a kind of sensitizing type aptamer test strips for detecting Salmonella, wherein sequence number 1 is PVC
Base plate, it is respectively arranged at two ends with sample pad 2 and adsorptive pads 5；The middle part of PVC base plate is provided with nitrocellulose filter detection layers 4,
Detection line 6 and nature controlling line 7 is had on it；Gold-marking binding pad 3 it is provided with between nitrocellulose filter detection layers 4 and sample pad 2.
Dendritic-gold particle-aptamer complex enhanced sensitivity probe it is coated with on described gold-marking binding pad 3；
Described dendritic is the dendritic of end sulfydryl；5 ' ends of described aptamer are by marking sulfhydryl, 3 ' end quilts
Biotin labeling.
It is coated with anti-salmonella monoclonal antibody on described detection line 6.
It is coated with Streptavidin on described nature controlling line 7.
Embodiment 2
A kind of for sensitizing type aptamer test strips detecting Salmonella and preparation method thereof, concrete operation step
As follows:
(1) preparation of dendritic-golden nanometer particle complex
The dendritic weighing 0.2mg is dissolved in 5mL acetone, takes the gold colloidal that newly synthesized particle diameter is 10nm molten
Liquid 1mL colloidal gold solution is distributed in 5mL pure water, under nitrogen protection and quickly stirring, is dropwise added drop-wise to by colloidal gold solution
In dendrimer solution, under magnetic agitation after room temperature reaction 12h, 10000rpm4 DEG C of centrifugal 30min, abandons supernatant, uses pure water
Washing precipitate, centrifuge washing and after being repeated 3 times by precipitation re-suspension liquid (20mM Na3PO4, 5%BSA, 0.25%Tween-
20,10% sucrose) resuspended, obtain 10 times of dendritic-golden nanometer particle complex solutions concentrated.
(2) preparation of dendritic-gold particle-aptamer complex
The Salmonella aptamer taking 10uL 1uM joins 2uL acetate buffer solution (0.5M, pH5.0) and 3uL
In 1mM tricarboxylic methyl acid phosphate solution, room temperature lucifuge places 1h, obtains the Salmonella aptamer solution of activation, Salmonella
Bacterium aptamer DNA sequence is: 5 '-SH-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG
ACA G-biotin-3’。
Take 10 times of dendritic-golden nanometer particle complex solution 200uL concentrated, be added dropwise to the sand of activation
In door Salmonella aptamer solution, under room temperature, stir 60min；The dATP taking 170uL 100uM joins in above-mentioned reactant liquor,
Stirring 30min is continued under room temperature；Progressively the NaCl solution of 0.1M is joined in reactant liquor, make the final concentration of NaCl reach
30mM, places after reacting 1h and hatch with 4 DEG C of refrigerator overnight under room temperature；It is resuspended extremely that 6500rpm is centrifuged 15min postprecipitation re-suspension liquid
200uL, obtains dendritic-gold particle-aptamer complex.
(3) preparation of dendritic-gold particle-aptamer enhanced sensitivity probe gold-marking binding pad
With three-dimensional planar point film gold spraying instrument by the dendritic-gold particle-aptamer complex of above-mentioned preparation
Uniformly spraying on glass fibre membrane, spouting liquid is 0.7uL/cm, is placed in 37 DEG C of drying post packages standby.
(4) preparation of coated film
With three-dimensional planar point film gold spraying instrument, the anti-salmonella monoclonal antibody solution that concentration is 1mg/mL is sprayed uniformly
It is coated onto on nitrocellulose filter, obtains detecting line (T line)；The solution of streptavidin that concentration is 1.5mg/mL is uniformly sprayed
On nitrocellulose filter, obtain nature controlling line (C line).
(5) preparation of sensitizing type Salmonella aptamer test strips
By sample film, dendritic-gold particle-aptamer enhanced sensitivity probe gold-marking binding pad, there is detection line
With on the nitrocellulose filter of nature controlling line and adsorptive pads paste composition successively to PVC base plate, the overlapping portion between adjacent paste
Divide a length of 2mm, be cut into the wide test strips of 3mm with cutting machine, obtain sensitizing type sramana based on dendroid nucleic acid enhanced sensitivity probe
Salmonella aptamer test strips, is loaded in aluminium foil bag sealing together with desiccant and saves backup.
Time specifically used, being taken out by the gold label test strip prepared, its mother glass fibrous membrane end is inserted into testing sample
In solution, take out after waiting 10min and with the naked eye observe.If nature controlling line 7 does not develops the color, then there are quality problems in test strips,
Testing result is invalid；If nature controlling line 7 and detection line 6 all develop the color, illustrate in testing sample containing object Salmonella, inspection
Survey line 6 develop the color the deepest then detection solution in Salmonella concentration the highest, testing sample be the positive；If detection line 6 does not develops the color, only
When having nature controlling line 7 to develop the color, illustrating not contain in testing sample Salmonella, testing sample is negative.
Claims (3)
1. for detecting a sensitizing type aptamer test strips for Salmonella, including PVC base plate (1), at the bottom of described PVC
Sample film (2), enhanced sensitivity probe gold-marking binding pad (3), nitrocellulose filter (4) and absorbing membrane (5) four it is pasted with successively on plate (1)
Plant paste；It is connected with each other between described adjacent paste and stacks；Described nitrocellulose filter is disposed with detection line on (4)
And nature controlling line (7) (6)；It is characterized in that, described gold-marking binding pad (3) is coated with dendritic-gold particle-nucleic acid and fits
Part nano-complex；Described dendritic is end sulfydryl dendritic；Described aptamer is can specificity
Identifying the aptamer of Salmonella, 5 ' ends of its DNA sequence are by marking sulfhydryl, and 3 ' ends are by biotin labeling；Described detection
Line is coated with anti-salmonella monoclonal antibody on (6)；Described nature controlling line is coated with Streptavidin on (7).
2. preparation test strips described in claim 1, it is characterised in that include following operating procedure:
(1) dendritic-golden nanometer particle complex is prepared
(2) dendritic-gold particle-adaptor complex is prepared
(3) dendritic-gold particle-adaptor complex prepared by step (2) is sprayed on glass fibre membrane, hydrojet
Amount is 0.7uL/cm, prepares gold-marking binding pad, dry for standby.
(4) spraying of the detection line position on nitrocellulose filter anti-salmonella monoclonal antibody solution, in nature controlling line position
Spraying Streptavidin, dry for standby.
(5) by sample film, gold-marking binding pad, there is detection line and the nitrocellulose filter of nature controlling line and adsorptive pads stickup group successively
Closing on PVC base plate, the length of overlapped part between adjacent paste is 2mm, is cut into the wide test strips of 3mm, with desiccant one
Rise to be packaged in aluminium foil bag and save backup.
3. method as claimed in claim 2, it is characterised in that in described step (1), the particle diameter of gold colloidal used is 10nm；Step
Suddenly be sprayed at described in (4) detection line position anti-salmonella monoclonal antibody be concentration be the anti-salmonella list of 1mg/mL
Clonal antibody solution；Be sprayed at described in step (4) Streptavidin of nature controlling line position be concentration be the strepto-of 1.5mg/mL
Avidin solution.
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| CN109900905A (en) * | 2019-02-28 | 2019-06-18 | 中国科学院广州生物医药与健康研究院 | A kind of colloidal gold strip and preparation method detecting carcinomebryonic antigen |
| CN110806477A (en) * | 2018-08-06 | 2020-02-18 | 国家纳米科学中心 | Pathogenic bacterium detection test strip, sensor and application thereof |
| CN117825685A (en) * | 2024-01-08 | 2024-04-05 | 云南省农业科学院质量标准与检测技术研究所 | Ochratoxin A colloidal gold marker and preparation method and application thereof |
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| CN106053805B (en) | 2018-09-21 |
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