CN106568951A - Nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip, and detection method - Google Patents
Nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip, and detection method Download PDFInfo
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- CN106568951A CN106568951A CN201610950378.7A CN201610950378A CN106568951A CN 106568951 A CN106568951 A CN 106568951A CN 201610950378 A CN201610950378 A CN 201610950378A CN 106568951 A CN106568951 A CN 106568951A
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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
- 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
Abstract
The invention provides a nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip. The nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip comprises a sample pad, a colloidal gold combined pad, a nitrocellulose membrane immobilized with streptavidin, and an absorbent pad; the nitrocellulose membrane is provided with a detection line and a quality control line; the sample pad, the colloidal gold combined pad, the nitrocellulose membrane, and the absorbent pad are glued onto a plastic liner board successively; colloidal gold labeled nucleic acid aptamer is arranged on the colloidal gold combined pad; a capture probe is immobilized on the detection line; and a quality control probe is immobilized on the quality control line. The invention also provides a method used for detecting escherichia coli O157:H7 with the nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip. According to the nucleic acid aptamer-based escherichia coli O157:H7 colloidal gold test strip, the escherichia coli O157:H7 outer membrane protein high specificity aptamer is taken as a recognition element of test strip instead of antibodies, so that problems of immunochromatographic test strip containing antibodies, such as high cost, large batch difference, induced cross reaction, and low sensitivity, are solved, and high-efficiency rapid detection of escherichia coli is realized.
Description
Technical field
The present invention relates to food-borne pathogenic microorganism detection technique field, specifically, is related to a kind of based on nucleic acid adaptation
The Escherichia coli O 157 of body：H7 colloidal gold strips and detection method.
Background technology
The alimentary toxicosis that food-borne pathogenic microorganism causes is one of important foodstuffs safety problem of global concern, according to the whole nation
Food origin disease monitoring information data shows that China is every year because the alimentary toxicosis number that food-borne pathogenic microorganism causes is accounted for
The 40-60% of all kinds of food origin disease subject populations.Escherichia coli O 157：It is to evaluate food peace in food-borne pathogens that H7 is
A kind of whether complete important indicator.Escherichia coli O 157 is directed in recent years：The detection method of H7 is from traditional loaded down with trivial details time-consuming
Long microorganism culturing, develops to the method for quick using technologies such as immunology, molecular biology and materials.This kind of method
By to the simple increasing bacterium of food samples, you can realize more sensitive, strong antijamming capability, high-throughout detection, it is adapted to scene sieve
Look into.
At present, for food-borne pathogens Fast Detection Technique application, domestic and international main flow is based on immunological technique
Sidestream chromatography reagent paper and enzyme linked immunological ELISA kit and PCR based on molecular biology method, although it is enzyme-linked at present to exempt from
Epidemic disease and the relatively large instrument of molecular biology method have many advantages in fast procuratorial organ face, but PCR methods and ELISA kit need specialty
Instrument realizing, and cost simple to operate does not need the immuno-chromatographic test paper strip of instrument more to meet basic unit and field quick detection
Requirement, it is considered to be solve the problems, such as the main path of food-borne microbial rapid detection.But due to the core of immunological technique
Antibody, it is low that test strips method equally exists sensitivity, it is impossible to quantitative, as a result by subjective judgment, high and artificial erroneous judgement of false positive etc.
Problem, still can not meet at present high sensitivity, meets field quick detection needs.Main reason is that based on antigen-antibody immunity
The Fast Detection Technique of principle, because of the limitation of antibody feature itself.The antigen-antibody immunocompetence site of invasive organism
Not clear, hybridoma screening blindness is big, and efficiency is low, and affinity of antibody is low, poor specificity；Cause existing detection technique sensitivity
The high problem of low, false positive rate；In addition, Antibody preparation depends on mammal, long preparation period, high cost, batch wise differences are big.
Therefore, high sensitivity high specificity, low cost, recognition component applied widely are found, and is increased with reference to novel nano-material
Detection sensitivity and substrate adaptability are the urgent needss of food-borne pathogenic microorganism quick detection development.
With the fast development of biotechnology, aptamer technology food safety detection, environmental contaminants monitoring with
And clinical diagnosises aspect has obtained good application.Aptamer essence is a class oligonucleotide sequence, specific by its
Three dimensional structure can show good with target high specific, combine high-affinity in the context of detection of mycotoxin
Application prospect.The following characteristics of aptamers so as to become the preferable antibody surrogate thing of toxic small molecule detection field：First, fit
Target molecule scope is wide, can be the small molecules such as toxin, metal ion, organic dyestuff, antibiotic, peptide, solves small molecule poison
Property target antibody is difficult to the problem for preparing；Two is to synthesize reproducible, low cost, only 1/6th of antibody, good stability；
Three be with reversible denaturation, can iii vitro chemical synthesis, room temperature storage and transport, the degeneration caused by temperature is reversible, is solved
Antibody generated time length, costly, the repeated problem with reproducibility difference；Four is to be easy to functionalization, and biochemical modification is realized
Signal generation, affinity capture, covalent cross-linking, nano-carrier modification, interface fixation etc., solve the problems, such as antibody labeling easy in inactivation；Most
Importantly, aptamers specificity is very good, the nuance in target molecule structure can be distinguished, such as theophylline and caffeine point
There was only the difference of hydrogen and methyl in minor structure, aptamers affinity difference just up to 10,000 times, therefore, aptamers can separate knot
Structure is similar or material of cross reaction, solves the problems, such as antibody cross reaction.Aptamers reagent paper is a kind of with aptamer knot
Close Lateral Flow Strip and the method for quick researched and developed, with simple to operate, stability is higher, is adapted to field quick detection etc. excellent
Point.
Although colibacillary adaptation body detecting method most of at present is all using the whole cell of escherichia coli as target
The aptamers that thing target sieving is obtained are element, and aptamers are poor to colibacillary identification specificity, in addition, bacterial reproduction
Hurry up, cell surface changes greatly, and aptamers recognition site is indefinite to easily lead to false positive, it is impossible to meet wanting for detection method
Ask.
The content of the invention
It is an object of the invention to provide a kind of Escherichia coli O 157 based on aptamer：H7 colloidal gold strips and its
Preparation method.
It is a further object of the present invention to provide a kind of detect Escherichia coli O 157 using above-mentioned colloidal gold strip：The side of H7
Method.
The present invention is based on following design：By Escherichia coli O 157：The quick increasing bacterium of H7, obtains sample, is loaded onto quick
On Test paper card, the principle of application " competition law ", Escherichia coli O 157：The outer membrane protein of H7 and the specificity of nano gold mark
Aptamer is combined, and is competed with aptamers complementation capture probe of the membrane marker in NC film detection line T line positions is drawn in advance.If
There is Escherichia coli O 157 in sample：H7, the then protein binding in nanometer gold-aptamer complex and sample, it is impossible to T
Complementary probe on line is combined, and detection line does not develop the color；Such as no Escherichia coli O 157 in sample：H7, nanometer gold-adaptation bluk recombination
Thing is combined with the complementary probe on T lines, the aggregation of nanogold particle and show obvious red line.By direct visual perception whether
There are T lines, so as to whether contain Escherichia coli O 157 in judgement sample：H7.
In order to realize the object of the invention, present invention firstly provides one kind can specific recognition Escherichia coli O 157：H7 adventitias
The aptamer of albumen, its nucleotides sequence is classified as 5 '-polyT-ATCCGTCACACCTGCTCTACGGCGCTCCCAACAGGCC
TCTCCTTACGGCATATTATGGTGTTGGCTCCCGTAT-3’.Preferably, the polyT sequences are：TTTTTTTTTTTT.
The present invention carries out O157 by paramagnetic particle method SELEX technologies：The screening of H7 outer membrane protein, detailed process is as follows：First
One is built by chemosynthesis about contain 1015Individual not homotactic single stranded oligonucleotide library, by surface O157 is fixed with：H7
The magnetic bead of outer membrane protein is incubated together with library and is fully combined with corresponding single-chain nucleic acid in order to target, then by Magneto separate
Program obtains the single nucleic acid strands combined with magnetic bead surfaces target specificity, followed by the combining Nucleic Acid Elution of elution program,
Finally enter performing PCR as template with the nucleic acid of these eluting to expand and for next round screening.By repeatedly combining, separating, wash
It is de- to eliminate that those are not combined with target, affinity is less or nucleic acid of middle affinity with amplification operating procedure, and there is high parent
Progressively it is enriched with the aptamer of power, is finally filtered out and O157：H7 outer membrane protein has the core of high specific adhesion
Sour aptamers.
The Escherichia coli O 157 based on aptamer of the present invention：H7 colloidal gold strips, including sample pad 1, colloid
Golden pad 2, the nitrocellulose filter 3 and absorption pad 4 that are fixed with Streptavidin, the nitric acid for being fixed with Streptavidin
Cellulose membrane 3 is provided with detection line 5 and nature controlling line 6, the sample pad 1, gold conjugation pad 2, is fixed with Streptavidin
Nitrocellulose filter 3 and absorption pad 4 are pasted onto successively on plastics lining board 7；
The gold conjugation pad 2 is fixed with the aptamer of the present invention of colloid gold label, and the detection line 5 is fixed with
Capture probe, the nature controlling line 6 is fixed with Quality Control probe.
The capture probe is：5’-ATACGGGAGCCAACACCATAATATGCCGTAAGGAGAGGCCTGTTGGGAGCG
CCGTAGAGCAGGTGTGACGGAT-biotin-3’(SEQ ID NO:2)。
Specifically, 5 ' ends of capture probe are the nucleotide sequence complementary with nucleic acid aptamer sequence of the present invention, and 3 ' is terminal modified
There is biotin.
The Quality Control probe is the polyA sequences of biotin labeling.Preferably, the Quality Control probe is：5’-
AAAAAAAAAAAA-biotin-3’(SEQ ID NO:3)。
Specifically, Quality Control probe is the polyA sequence complementary with nucleic acid aptamer sequence polyT of the present invention, and 3 ' is terminal modified
There is biotin.
The sample pad 1, gold conjugation pad 2, be fixed with the nitrocellulose filter 3 and absorption pad 4 of Streptavidin according to
The secondary spacing interaction cascading with 2mm or so is pasted on plastics lining board 7, and the test strips width is 4mm or so.
Preferably, the material of sample pad of the present invention is cellulose membrane, and the material of the gold conjugation pad is glass
Fibrous membrane.
The colloidal gold strip of the present invention can be prepared as follows, comprise the following steps：
The preparation of S1, gold nano grain；
The preparation of S2, gold nano grain-aptamer complex；
The preparation of S3, detection line (T lines) and nature controlling line (C lines)；
The assembling of S4, colloidal gold strip.
S1 is specially：Take 1% HAuCl4Aqueous solution 1mL, adds 99mL distilled water, ebuillition of heated to add thereto rapidly
Enter the sodium citrate 5mL that concentration is 1%, be stirred vigorously, solution colour gradually becomes red by colourless.Continue to heat after 15min
Stop heating, and continue to stir to room temperature, containing the gold nano grain that particle diameter is 15 ± 2nm in the Au NPs stock solutions for obtaining, 4 DEG C
Keep in dark place.
S2 is comprised the following steps：
S21, take 6 μ L, 100 μM of sulfydryls and be adapted to liquid solutions, add hydrochloric acid three (2- carboxyethyls) the phosphine solution of 6 μ L 5mg/mL,
4 DEG C of activation 2h；Wherein, the sulfydryl aptamers refer to 5 ' ends of the nucleic acid aptamer sequence with a mercapto groups；
S22, take Au NPs stock solutions 5mL 15min is centrifuged with 13000rmp, remove supernatant and simultaneously add the ultra-pure water of 5mL to answer
It is molten, the sulfydryl aptamers after activating then are added thereto to, mix, 16h is reacted at room temperature；Then, add 50 μ L's 1%
Sodium dodecyl sulfate solution reacts at room temperature 1h to final concentration of 0.01%；Then, (such as 8 times) addition several times
24h is reacted at room temperature in 2mol/L NaCl solutions totally 80 μ L to the final concentration of 160mM of salt ion, continuation；
S23, the solution obtained in S22 is removed into unreacted sulfydryl aptamers with 13000rmp centrifugation 15min, and with surpassing
Pure water is cleaned once, and in being dissolved in buffer, 4 DEG C keep in dark place, and obtain final product the aptamer fixative of colloid gold label, fixed
Contain the gold nano grain-aptamer complex in liquid.
Wherein, buffer described in S23 is 2% sucrose containing 0.5%PEG, 0.1% tween 20,0.02% magnesium sulfate,
The pH 7.4 of 0.05% ammonium sulfate and 1% ovalbumin, the phosphate buffer of 10mM.
S3 is comprised the following steps：
The preparation of S31, the capture probe of biotin labeling；
The preparation of S32, the Quality Control probe of biotin labeling：
The preparation of S33, detection line and nature controlling line：By 10-35 μ L 1mg/mL avidin solutions respectively with 10 μM of 10-35 μ L
The capture probe and Quality Control probe of biotin labeling is incubated 2h at 4 DEG C, and using film instrument Deca is drawn Streptavidin is being fixed with
Detection line and nature controlling line are respectively obtained on nitrocellulose filter.
S4 is specially：By the sample pad, gold conjugation pad, the nitrocellulose filter for being fixed with Streptavidin and suction
Receive pad to be pasted onto successively on plastics lining board；Amount Deca by the fixative with 5 μ L per bar on gold conjugation pad, in room temperature
5min is air-dried down, and 4 DEG C keep in dark place, and obtain final product the colloidal gold strip.
In the present invention, the preparation method for being fixed with the nitrocellulose filter of Streptavidin is as follows：By 1mg/mL strepto-s parent
1 μ L/cm are pressed with plain solution2Amount with draw a film instrument be sprayed onto on nitrocellulose filter, be fixed on film by physical absorption.The mercapto
The capture probe and Quality Control probe of base aptamers and biomarker is by Shanghai life work synthesis.
The present invention is also provided and detects Escherichia coli O 157 using the colloidal gold strip：The method of H7, including sample increasing
The step of bacterium culture, ELISA test strip and result interpretation；Concrete grammar is as follows：
(1) 23.5g brain heart infusion broths (BHI) culture medium is weighed, addition is preheated to 42 ± 1 DEG C of sterilized water 1L, dissolves standby
With；
(2) sterile working takes 25g or 25mL samples in homogenizing bag, and is added thereto to the brain heart leaching after 225mL preheatings
Liquid broth bouillon, is put into homogenizing 2min in homogenizer, and 42 DEG C stand or shaken cultivation 8h；
(3) (2) gained culture is mixed, takes 1mL and be added in test tube, 100 DEG C of heating 5min are cooled to room temperature, use
Dropper Deca 3-4 drop (about 100 μ L) reacts 5-8min, sentence read result, more than 30min colour developing nothings to the sample pad of test strips
Effect；
(4) result interpretation：
Negative reaction：Nature controlling line, detection line develop the color；
Positive reaction：Nature controlling line develops the color, and detection line does not develop the color；
Failure reaction：If nature controlling line does not develop the color, detection failure or test strips fail.
The method that the present invention is provided is applied to Escherichia coli O 157：H7 quick detections, are capable of achieving escherichia coli in sample
O157：H7 detections in most short 8 hours, detection sensitivity 1CFU/25g.The demand of daily bread monitoring can be met, be adapted to food sample
Escherichia coli O 157 in product, Environmental Water and fresh and live agricultural product：The detection of H7.
The quick enriched medium that the method that the present invention is provided has extremely strong substrate adaptability, sample is capable of achieving large intestine bar
Bacterium O157：The fast breeding of H7, while suppressing the growth of infectious bacteria, can avoid the interference of a large amount of other factors, beneficial to sample
The rapid screening of product.
In order to reach expected sensitivity and accuracy, the present invention builds detection using the aptamer of nano gold mark
System, in the reaction system, with Escherichia coli O 157：H7 outer membrane protein specific nucleic acids aptamers substitute antibody, it is to avoid
Antigen-antibody immunocompetence site fails to understand that hybridoma screening blindness is big, and efficiency is low, and affinity of antibody is low, poor specificity；Cause
The problem that existing detection technique sensitivity is low, false positive rate is high；In addition, the production cost of aptamers be only antibody six/
One, can synthesize in batches in vitro, this recognition component overcomes Antibody preparation cycle length, high cost, the big shortcoming of batch wise differences,
The stability of ELISA test strip is enhanced, cost is reduced, and increases shelf life.In addition, Escherichia coli O 157：H7 adventitia eggs
White specific nucleic acid aptamers on the aptamers specificity and stability with whole somatic cells as target compared to having excellent
Gesture, therefore, the E. coli detection method that the present invention is provided applies sensitivity height, high specificity, low cost, applied widely
Recognition component, and increase detection sensitivity and substrate adaptability with reference to novel nano-material, the micro- life of food-borne pathogenic can be met
The demand of thing quick detection.
This method is using for Escherichia coli O 157：The specificity aptamers of stable outer membrane protein are expressed outside H7 cell membrane
As the recognition component of chromatographic test paper, sensitivity and the specificity of detection are improved, realize efficient, simple, accurate quick detection,
There is good prospect in the highly sensitive quick analysis field of food-borne pathogens, so as to find pollution in time, it is to avoid harm, effectively
Reduce Escherichia coli O 157：The harm of H7, protects the health of the mankind.
Description of the drawings
Fig. 1 is Escherichia coli O 157 of the embodiment of the present invention 1 based on aptamer：The structure of H7 colloidal gold strips is shown
It is intended to.Wherein, 1- sample pad, 2- gold conjugation pads, 3- nitrocellulose filters, 4- absorption pads, 5- detection lines, 6- nature controlling lines,
7- liner plates, 8- gold nano grains-aptamer complex, 9- capture probes, 10- Quality Control probes, 11- Avidins.
Fig. 2 is colloidal gold strip operation principle schematic diagram in the embodiment of the present invention 2.
Fig. 3 is colloidal gold strip testing result process decision chart in the embodiment of the present invention 2.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, it is raw materials used to be commercial goods.
The percentage sign " % " being related in the present invention, if not specified, refers to mass percent；But the percentage of solution
Than unless otherwise specified, referring to the grams containing solute in 100ml solution.
Escherichia coli O 157 of the embodiment 1 based on aptamer：H7 colloidal gold strips and preparation method thereof
The present embodiment provides a kind of Escherichia coli O 157 based on aptamer：H7 colloidal gold strip quick detection bodies
System, the detection system includes can specific recognition E.coli O157：High-affinity aptamers of H7 outer membrane protein and with suitable
Part is the colloidal gold strip of recognition component.
The Escherichia coli O 157 based on aptamer：H7 colloidal gold strips, including sample pad 1, gold colloidal knot
Close pad 2, be fixed with the nitrocellulose filter 3 and absorption pad 4 of Streptavidin, the cellulose nitrate for being fixed with Streptavidin
Plain film 3 is provided with detection line 5 and nature controlling line 6, the sample pad 1, gold conjugation pad 2, the nitric acid for being fixed with Streptavidin
Cellulose membrane 3 and absorption pad 4 are pasted onto successively on plastics lining board 7；
The gold conjugation pad 2 is fixed with the aptamer of colloid gold label, and the detection line 5 is fixed with catches
Probe is obtained, the nature controlling line 6 is fixed with Quality Control probe.
The aptamer is can specific recognition Escherichia coli O 157：The aptamer of H7 outer membrane protein, its core
Nucleotide sequence such as SEQ ID NO:Shown in 1.
The capture probe is：5’-ATACGGGAGCCAACACCATAATATGCCGTAAGGAGAGGCCTGTTGGGAGCG
CCGTAGAGCAGGTGTGACGGAT-biotin-3’(SEQ ID NO:2)。
Specifically, 5 ' ends of capture probe are the nucleotide sequence complementary with nucleic acid aptamer sequence of the present invention, and 3 ' is terminal modified
There is biotin.
The Quality Control probe is the polyA sequences of biotin labeling.Preferably, the polyA sequences are：5’-
AAAAAAAAAAAA-biotin-3’(SEQ ID NO:3)。
Specifically, Quality Control probe is the polyA sequence complementary with polyT in nucleic acid aptamer sequence of the present invention, and 3 ' ends are repaiied
It is decorated with biotin.
The sample pad 1, gold conjugation pad 2, be fixed with the nitrocellulose filter 3 and absorption pad 4 of Streptavidin according to
The secondary spacing interaction cascading with 2mm or so is pasted on plastics lining board 7, and the test strips width is 4mm or so.
Preferably, the material of the sample pad is cellulose membrane, and the material of the gold conjugation pad is glass fibre membrane.
The colloidal gold strip of the present invention can be prepared as follows：Comprise the following steps：
The preparation of S1, gold nano grain；
The preparation of S2, gold nano grain-aptamer complex；
The preparation of S3, detection line (T lines) and nature controlling line (C lines)；
The assembling of S4, colloidal gold strip.
S1 is specially：Take 1% HAuCl4Aqueous solution 1mL, adds 99mL distilled water, ebuillition of heated to add thereto rapidly
Enter the sodium citrate 5mL that concentration is 1%, be stirred vigorously, solution colour gradually becomes red by colourless.Continue to heat after 15min
Stop heating, and continue to stir to room temperature, containing the gold nano grain that particle diameter is 15 ± 2nm in the Au NPs stock solutions for obtaining, 4 DEG C
Keep in dark place.
S2 is comprised the following steps：
S21, take 6 μ L, 100 μM of sulfydryls and be adapted to liquid solutions, add hydrochloric acid three (2- carboxyethyls) the phosphine solution of 6 μ L 5mg/mL,
4 DEG C of activation 2h；Wherein, the sulfydryl aptamers refer to 5 ' ends of the nucleic acid aptamer sequence with a mercapto groups；
S22, take Au NPs stock solutions 5mL 15min is centrifuged with 13000rmp, remove supernatant and simultaneously add the ultra-pure water of 5mL to answer
It is molten, the sulfydryl aptamers after activating then are added thereto to, mix, 16h is reacted at room temperature；Then, add 50 μ L's 1%
Sodium dodecyl sulfate solution reacts at room temperature 1h to final concentration of 0.01%；Then, (such as 8 times) addition several times
24h is reacted at room temperature in 2mol/L NaCl solutions totally 80 μ L to the final concentration of 160mM of salt ion, continuation；
S23, the solution obtained in S22 is removed into unreacted sulfydryl aptamers with 13000rmp centrifugation 15min, and with surpassing
Pure water is cleaned once, and in being dissolved in buffer, 4 DEG C keep in dark place, and obtain final product the aptamer fixative of colloid gold label, fixed
Contain the gold nano grain-aptamer complex in liquid.
Wherein, buffer described in S23 is 2% sucrose containing 0.5%PEG, 0.1% tween 20,0.02% magnesium sulfate,
The pH 7.4 of 0.05% ammonium sulfate and 1% ovalbumin, the phosphate buffer of 10mM.
S3 is comprised the following steps：
The preparation of S31, the capture probe of biotin labeling；
The preparation of S32, the Quality Control probe of biotin labeling；
The preparation of S33, capture probe and Quality Control probe storing solution：By 10-35 μ L 1mg/mL avidin solutions respectively with
The capture probe and Quality Control probe of 10-35 μ 10 μM of biotin labelings of L is incubated 2h at 4 DEG C, is being fixed with using film instrument Deca is drawn
Detection line and nature controlling line are respectively obtained on the nitrocellulose filter of Streptavidin.
S4 is specially：By the sample pad, gold conjugation pad, the nitrocellulose filter for being fixed with Streptavidin and suction
Receive pad to be pasted onto successively on plastics lining board；Amount Deca by the fixative with 5 μ L per bar on gold conjugation pad, in room temperature
5min is air-dried down, and 4 DEG C keep in dark place, and obtain final product the colloidal gold strip, and structure is as shown in Figure 1.
Embodiment 2 detects Escherichia coli O 157 using colloidal gold strip：The method of H7
The present embodiment provides a kind of method for fast detecting colibacillus based on aptamer, and methods described is with E.coli
O157：H7 outer membrane protein specifically binds aptamer as the identification molecule of colloidal gold strip, using nanometer gold as Jie
Matter, using in Sidestream chromatography principle detection sample whether there is escherichia coli.
The colloidal gold strip detection Escherichia coli O 157 prepared using embodiment 1：The operation principle of H7 is as shown in Figure 2.
Concrete grammar is as follows：
(1) 23.5g brain heart infusion broths (BHI) culture medium is weighed, addition is preheated to 42 ± 1 DEG C of sterilized water 1L, dissolves standby
With；
(2) sterile working takes 25g or 25mL samples in homogenizing bag, and is added thereto to the brain heart leaching after 225mL preheatings
Liquid broth bouillon, is put into homogenizing 2min in homogenizer, and 42 DEG C stand or shaken cultivation 8h；
(3) (2) gained culture is mixed, takes 1mL and be added in test tube, 100 DEG C of heating 5min are cooled to room temperature, use
Dropper Deca 3-4 drop (about 100 μ L) reacts 5-8min, sentence read result, more than 30min colour developing nothings to the sample pad of test strips
Effect；
(4) result judgement：See Fig. 3.
Negative reaction：Nature controlling line, detection line develop the color；
Positive reaction：Nature controlling line develops the color, and detection line does not develop the color；
Failure reaction：If nature controlling line does not develop the color, detection failure or test strips fail.
The present invention replaces antibody as the recognition component of Sidestream chromatography test strips with escherichia coli high specific aptamers, gram
Take and relied on the immuno-chromatographic test paper strip antibody high cost of antibody, batch wise differences are big, there is cross reaction, sensitivity is not high have been lacked
Point, and the cost of detection can be substantially reduced, realize efficient, simple and direct, the inexpensive quick detection of escherichia coli.Using we
Method is capable of achieving Escherichia coli O 157 in sample：H7 detections in most short 8 hours, detection sensitivity 1CFU/25g.Daily bread can be met
The demand of monitoring, is adapted to Escherichia coli O 157 in food samples, Environmental Water and fresh and live agricultural product：The detection of H7.
Although above with a general description of the specific embodiments the present invention is described in detail,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, without departing from theon the basis of the spirit of the present invention these modifications or improvements, belong to the scope of protection of present invention.
Sequence table
<110>Beijing Research Center For Agricultural Standards and Testing
<120>Escherichia coli O 157 based on aptamer：H7 colloidal gold strips and detection method
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Claims (10)
1. specific recognition Escherichia coli O 157：The aptamer of H7 outer membrane protein, it is characterised in that its nucleotides sequence is classified as
5’-polyT-ATCCGTCACACCTGCTCTACGGCGCTCCCAACAGGCCTCTCCTTACGGCATATTATGGTGTTGGCTCC
CGTAT-3’；Preferably, the polyT sequences are：TTTTTTTTTTTT.
2. the Escherichia coli O 157 of aptamer is based on：H7 colloidal gold strips, it is characterised in that including sample pad (1), glue
Body gold pad (2), the nitrocellulose filter (3) and absorption pad (4) of Streptavidin are fixed with, described to be fixed with strepto- affine
The nitrocellulose filter (3) of element is provided with detection line (5) and nature controlling line (6), the sample pad (1), gold conjugation pad (2),
It is fixed with the nitrocellulose filter of Streptavidin (3), absorption pad (4) to be pasted onto successively on plastics lining board (7)；
The gold conjugation pad (2) is fixed with aptamer described in the claim 1 of colloid gold label, the detection line
(5) capture probe is fixed with, the nature controlling line (6) is fixed with Quality Control probe；
The capture probe is：5’-ATACGGGAGCCAACACCATAATATGCCGTAAGGAGAGGCCTGTTGGGAGCGCCGT
AGAGCAGGTGTGACGGAT-biotin-3’；
The Quality Control probe is the polyA sequences of biotin labeling；Preferably, the Quality Control probe is：5’-AAAAAAAAAAAA-
biotin-3’。
3. colloidal gold strip according to claim 2, it is characterised in that the sample pad (1), gold conjugation pad
(2) nitrocellulose filter (3), the absorption pad (4) for, being fixed with Streptavidin is pasted successively with the spacing interaction cascading of 2mm
On plastics lining board (7), the test strips width is 4mm.
4. the colloidal gold strip according to Claims 2 or 3, it is characterised in that the material of the sample pad is cellulose
Film, the material of the gold conjugation pad is glass fibre membrane.
5. the preparation method of colloidal gold strip described in any one of claim 2-4, it is characterised in that comprise the following steps：
The preparation of S1, gold nano grain；
The preparation of S2, gold nano grain-aptamer complex；
The preparation of S3, detection line and nature controlling line；
The assembling of S4, colloidal gold strip.
6. method according to claim 5, it is characterised in that S1 is specially：Take 1% HAuCl4Aqueous solution 1mL, adds
99mL distilled water, ebuillition of heated is added thereto to rapidly the sodium citrate 5mL that concentration is 1%, stirring, stops after heating 15min
Heating, and continue to stir to room temperature, containing the gold nano grain that particle diameter is 15 ± 2nm, 4 DEG C of lucifuges in the Au NPs stock solutions for obtaining
Preserve.
7. the method according to claim 5 or 6, it is characterised in that S2 is comprised the following steps：
S21, take 6 μ L, 100 μM of sulfydryls and be adapted to liquid solutions, add hydrochloric acid three (2- carboxyethyls) the phosphine solution of 6 μ L 5mg/mL, 4 DEG C
Activation 2h；
S22, take Au NPs stock solutions 5mL 15min is centrifuged with 13000rmp, remove supernatant and simultaneously add 5mL water to redissolve, then to it
The middle sulfydryl aptamers added after activation, mix, and 16h is reacted at room temperature；Then, the lauryl sulphate acid of 50 μ L 1% is added
Sodium solution reacts at room temperature 1h to final concentration of 0.01%；Then, 2mol/L NaCl solutions are added several times, and totally 80 μ L are extremely
24h is reacted at room temperature in the final concentration of 160mM of salt ion, continuation；
S23, the solution obtained in S22 is removed into unreacted sulfydryl aptamers with 13000rmp centrifugation 15min, and cleaned with water
Once, it is dissolved in buffer, 4 DEG C keep in dark place, obtains final product the aptamer fixative of colloid gold label, contains in fixative
The gold nano grain-aptamer complex；
Wherein, buffer described in S23 is 2% sucrose containing 0.5%PEG, 0.1% tween 20,0.02% magnesium sulfate,
The pH 7.4 of 0.05% ammonium sulfate and 1% ovalbumin, the phosphate buffer of 10mM.
8. the method according to any one of claim 5-7, it is characterised in that S3 is comprised the following steps：
The preparation of S31, the capture probe of biotin labeling；
The preparation of S32, the Quality Control probe of biotin labeling；
The preparation of S33, detection line and nature controlling line：By the avidin solution of 10-35 μ L 1mg/mL respectively with 10 μM of lifes of 10-35 μ L
The capture probe and Quality Control probe of thing element labelling is incubated 2h at 4 DEG C, and using film instrument Deca is drawn the nitre of Streptavidin is being fixed with
Detection line and nature controlling line are respectively obtained on acid cellulose film.
9. the method according to claim 7 or 8, it is characterised in that S4 is specially：The sample pad, gold colloidal are combined
The nitrocellulose filter and absorption pad for padding, being fixed with Streptavidin is pasted onto successively on plastics lining board；By the fixative with 5
Amount Deca of the μ L per bar air-dries 5min on gold conjugation pad, at room temperature, and 4 DEG C keep in dark place, and obtains final product the gold colloidal examination
Paper slip.
10. colloidal gold strip detection Escherichia coli O 157 described in any one of claim 2-4 is utilized：The method of H7, its feature
It is, including the step of sample Zengjing Granule, ELISA test strip and result interpretation；Concrete grammar is as follows：
(1) 23.5g brain heart infusion broth culture medium is weighed, addition is preheated to 42 ± 1 DEG C of sterilized water 1L, dissolves standby；
(2) sterile working takes 25g or 25mL samples in homogenizing bag, and the brain heart infusion meat being added thereto to after 225mL preheatings
Soup culture medium, is put into homogenizing 2min in homogenizer, and 42 DEG C stand or shaken cultivation 8h；
(3) (2) gained culture is mixed, takes 1mL and be added in test tube, 100 DEG C of heating 5min are cooled to room temperature, use dropper
Deca 3-4 is dropped in the sample pad of test strips, reacts 5-8min, and sentence read result is invalid more than 30min colour developings；
(4) result interpretation：
Negative reaction：Nature controlling line, detection line develop the color；
Positive reaction：Nature controlling line develops the color, and detection line does not develop the color；
Failure reaction：If nature controlling line does not develop the color, detection failure or test strips fail.
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