CN102565389A - Nano/ALISA method and kit used for rapid detection of Salmonella - Google Patents
Nano/ALISA method and kit used for rapid detection of Salmonella Download PDFInfo
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- CN102565389A CN102565389A CN2011103156732A CN201110315673A CN102565389A CN 102565389 A CN102565389 A CN 102565389A CN 2011103156732 A CN2011103156732 A CN 2011103156732A CN 201110315673 A CN201110315673 A CN 201110315673A CN 102565389 A CN102565389 A CN 102565389A
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Abstract
The invention relates to a novel method used for rapid detection of Salmonella based on nano/aptamer-linked immobilized sorbent assay (Nano/ALISA) and a kit used for rapid detection of Salmonella, and the method is established by using an aptamer to substitute an antibody for capture of Salmonella and using gold nanoparticle couplet as a signal reporting group. The method comprises the following steps: (1) modifying the surface of magnetic particles with a specific aptamer of Salmonella; (2) adding a specimen for a reaction; (3) adding a specific primary antibody of Salmonella for reaction; (4) adding the couplet of secondary antibody-gold nanoparticle-reporting group for reaction; and (5) adding a specific substrate for reaction or directly recording light signals at the time. The kit comprises (1) the magnetic particles modified by the specific aptamer, (2) the specific primary antibody of Salmonella, (3) the couplet of secondary antibody-gold nanoparticle-reporting group, (4) confining liquid, (5) rinsing liquid, (6) the substrate, (7) positive control bacteria liquid and negative control bacteria liquid, etc.
Description
Technical field
The invention belongs to technical field of biological; Relate to utilize aptamers to replace antibody to carry out that detection of Salmonella is caught and with the nm of gold couplet as the signal reporter group; Be particularly related to use that this technology sets up a kind of based on nanometer/aptamers combined immunization absorption method (nano/aptamer-linked immobilized sorbent assay, novel detection of Salmonella method for quick Nano/ALISA) and be used for the kit of detection of Salmonella fast detecting.
Background technology
Detection of Salmonella (Salmonella) is common infecting both domestic animals and human cause of disease bacterium, extensively is present in the enteron aisle of all kinds of animals, and by in the water and soil earth of animal fecal pollution, can causes enteritis, enteric fever even septicemia etc.Its infection morbidity accounts for the main status of food posioning, is listed in one of Category B notifiable disease by China's Law on the Prevention and Control of Infectious Diseases by its typhoid that causes, all remains serious public health problem in developing country and developed country.The Salmonella infection of the annual report of the U.S. has 40,000 examples approximately, but because many infecteds do not seek medical advice and report, actual the infected's number is more than 20 times of this numeral." the peanut butter poisoning " that in September, 2008, the U.S. took place to be caused by Salmonella typhimurtum; This is the U.S.'s the most serious salmonellosis epidemic situation over 10 years; Cause food maximum on the American history to recall incident, and forced the Obama administration existing food security method is revised.In China, particularly vast rural basic level, because of salmonellal infection happens occasionally, its velocity of propagation is fast, it is wide to involve, in case take place, is easy to cause that diffusion is popular.Zhang Su etc. were to 1985～2000 China's food poisoning situation selective analysis, and in the number of the infected of microorganism property food poisoning, detection of Salmonella is the most serious; Wang Shijie etc. show that to the analysis result of 766 food posionings of 1994～2003 China detection of Salmonella occupies second.Detection of Salmonella constitutes a threat to human beings'health; And bring heavy financial burden to country; The prevention that event breaks out detection of Salmonella; And to the making a definite diagnosis fast and suspicious patient is investigated of the first routine case, accomplish discovery early, early treatment, early isolate, search fast the infection sources, to cut off route of transmission etc. very important to controlling this disease.
The laboratory of detection of Salmonella detection at present is main foundation to detect pathogen still, and its trace routine comprises collection of specimens inoculation, separation and Culture, biochemical identification, and a plurality of links such as serological typing take four to eight days at the soonest and just can make the final inspection diagnosis.In recent years, the scholar has launched a large amount of research work to the Fast Detection Technique of detection of Salmonella all over the world, utilizes special biological identification element to carry out quick diagnosis, like molecular biology and immunology detection technology etc.(polymerase chain reaction PCR) can increase the inhereditary material of trace 1,000,000 times rapidly, makes the various analyzing and testing projects that originally can't carry out be able to implement through polymerase chain reaction.But PCR often needs to extract earlier the DNA or the RNA of detection of Salmonella, utilizes the special primer of having reported to increase, and can carry out qualitative or detection by quantitative according to amplification, can detect＜detection of Salmonella of 100CFU.Though advantages such as PCR has fast, sensitive, special detecting aspect the detection of Salmonella still defectiveness, are operated like the needs professional, used reagent and instrument costliness, the testing result false positive is high, all is the difficult problem that researchers face.The immunology detection technology then utilizes the specific reaction between antigen and the antibody to realize the fast detecting of detection of Salmonella, and the most frequently used is that (enzyme linked immunosorbent assay, ELISA), the detection sensitivity of having reported is 10 to the enzyme linked immunological adsorption technology
5CFU ml
-1Elisa technique since occurring the seventies in 20th century, just because of its specificity is high, susceptibility is strong, simple quick, do not need expensive instrument and equipment, be particularly suitable for advantage such as the screening work of great amount of samples is received much concern.In addition, immune magnetic separates (immunomagnetic separation) technology can catch special thalline in various samples, play concentrated effect, often with the elisa technique coupling.But the sensitivity of elisa technique and specificity often depend on the size of affinity between antibody and the antigen, and antibody has shortcomings such as preparation difficulty, mark are loaded down with trivial details, easy sex change, and this all is that we need improved keys; In addition, the target that ELISA detects is difficult to increase through PCR as nucleic acid is direct, has received considerable restraint so signal amplifies efficient, and detection of Salmonella often concentration is lower in actual sample.Therefore, press for new easy, quick, the sensitive method of development and satisfy detection detection of Salmonella.
Aptamers (aptamer) be one type have high affinity with can high degree of specificity the oligonucleotide sequence of identification combining target molecule, its target molecule scope is wide, from micromolecule, like medicine and dyestuff; To complicated biomacromolecule, like the enzyme molecule, polypeptide and protein, even complete cell.This artificial receptors is through a kind of new in-vitro screening technology-index concentration Fas lignand system evolution (systematic evolution of ligands by exponential enrichment; What SELEX), screening obtained from single stranded oligonucleotide library at random.Because aptamers has identical with antibody or is superior to the selectivity and the affinity of antibody, so can replace antibody as a kind of novel molecular recognition component.In addition, compare with antibody, aptamers also has several advantages, as thermal stability increase, the anti-value of pH widely and salinity and synthetic, modification and curing be easy.Therefore, aptamers and ELISA method is combined into for a new development trend.2006, Vivekananda etc. proposed aptamers combined immunization absorption method (aptamer-linked immobilized sorbent assay, ALISA) this notion, and successfully detected francisella tularensis first in article.
Nano material (nanomaterials) is meant in three dimensions, to have at least one dimension to be in nanoscale scope (1～100nm) or the material with property that is made up of as elementary cell them.Because the small size of nano material and special surface state, make it show many characteristics that not only had been different from microscopic particle but also had been different from macro object, for example quantum size effect, surface effect, small-size effect and macro quanta tunnel effect etc.The fast development of nanometer technology is that the signal amplification mode of biological detection provides new selection, and both combinations have become new development trend.In field of biological detection; Utilize the great specific surface area of nano material; Researchers can make up the couplet of various biomolecules more easily; Can when loading Target Recognition molecule (like oligonucleotide probe, antibody etc.), carry a large amount of signaling molecules (as producing the protein of colour developing or fluorescence signal) and be used for signal and strengthen, this just for the highly sensitive biological detection method of development provides maybe.
Wherein, Nm of gold (gold nanoparticles; AuNPs) because of it has excellent optical property, electrical properties, chemical activity, bio-compatibility, make its application the most extensive, can trace back to the application of the seventies immunity gold in last century in bio-imaging the earliest at biological field.Nm of gold also has the unique biological compatibility, can be widely used in the mark of materials such as DNA, antibody, antigen and enzyme.In recent years, domestic and international research mechanism has reported in succession and has utilized nm of gold to carry out the Biological Detection strategy that signal amplifies for a plurality of biomolecule of carrier coupling.Since 2003, Mirkin seminar has reported a series of high sensitivity Protein Detection strategies based on " antibody-nm of gold-bar code DNA " coupling structure, and a large amount of bar code DNA as signaling molecule that utilize the nm of gold surface to load strengthen detection signal greatly.2009, Chun-Ping Jia etc. was assembled in the nano Au particle surface altogether with p53 antibody and Avidin HRP, is prepared into p53 antibody-nm of gold-Avidin HRP composite nanostructure, can detect 5pg ml in the 2h
-1P53, reduced by 25 times than the LDL of traditional E LISA.
Summary of the invention
First technical matters that the present invention will solve provides the special aptamers mark magnetic-particle that is used to catch detection of Salmonella.
The immunity magnetic separation technique can be caught special thalline in various samples, play concentrated effect; Aptamers can not rely on living animal or living cells through the solid phase synthesis mass preparation of robotization, and error is little between batch, and good reproducibility is with low cost, and good stability, and sex change is reversible, is easy to carry out biological chemistry and modifies.Therefore, the present invention through chemistry or biological method with special aptamers of detection of Salmonella and magnetic-particle coupling.For the aptamers that makes the magnetic-particle surface keeps setting rather than lodging on the magnetic-particle surface as far as possible, be beneficial to it and catch detection of Salmonella in this step, select for use spacer DNA (spacer DNA) to seal the vacant site of magnetic-particle, be generally 20 T or A; Correspondingly also design continuous identical base, improve the efficient that aptamers is caught detection of Salmonella at aptamers mark end.The aptamers that has specific recognition detection of Salmonella function among the present invention is not limited to a kind of concrete aptamers, and comprises that all can modify the special aptamers of magnetic-particle surface detection of Salmonella through suitable method.Method of modifying such as glutaraldehyde cross-linking method, carbodiimide method, Avidin-biotin method etc.
Second technical matters that the present invention will solve provides and is used for the nm of gold couplet that detection of Salmonella detects.Because great specific surface area of nm of gold and excellent biological compatibility; We can make up the couplet of various biomolecules easily, can when loading Target Recognition molecule (like the special aptamers of detection of Salmonella, antibody etc.), carry a large amount of signaling molecules (like peroxidase, alkaline phosphatase etc.) and be used for signal and strengthen.Therefore nm of gold couplet of the present invention can be used as a kind of signal and amplifies reagent, and substrate reactions or id reaction through catalyst play the effect that signal amplifies.
According to the present invention, nm of gold is connected with existing method with the coupling of Target Recognition molecule, signaling molecule.Wherein, the aptamers sequence modification has sulfydryl, is connected through golden sulfide linkage with nm of gold.Usually with the centrifugal nano-Au solution that obtains about 10nM that concentrates of former nano-Au solution, be placed on 4 ℃ and descend subsequent use; The sulfhydrylation aptamers is added the nano-Au solution mixing spend the night (16h), aging with salt then, room temperature is placed 40h, and then centrifuge washing is 3 times, suspends 4 ℃ of preservations with the reactant liquor of using in testing at last.And protein can be at an easy rate through multiple acting force and nano Au particle couplings such as golden sulfide linkage, electrostatic force.The pH value of regulating nano-Au solution usually adds 37 ℃ of vibrations of protein solution and hatched 4 ℃ of hold over night 10 minutes a little more than isoelectric points of proteins; Add 5%PEG to final concentration 0.5%, mixing was placed 1 hour for 4 ℃; Centrifuge washing 3 times, resuspended with storage solutions at last.Described storing solution preferably contains 0.5%BSA, 2.5% sucrose, 0.1%PEG, the PBS damping fluid of pH 7.4.Can use 0.1M K when in addition, need improving the pH value of nm of gold
2CO
3, can use 0.1M HCl when need reducing the pH value of nm of gold.
First purpose of the present invention is to disclose a kind of novel immunology detection technology (Nano/ALISA), and utilizes this technology to carry out the method for fast detecting detection of Salmonella.This new method can may further comprise the steps:
1. catch detection of Salmonella with the special aptamers of detection of Salmonella, carry out the separation and concentration detection of Salmonella through magnetic-particle;
2. special one anti-reaction that adds detection of Salmonella;
3. add the nm of gold couplet of the invention described above, obtain to be applicable to high sensitivity immune detection " sandwich " structure;
4. generate certain color or fluorescence and detect through can produce colour developing or the protein catalytic substrate of fluorescence signal on the nm of gold couplet.
Second purpose of the present invention provides a kind of detection of Salmonella quick detection kit based on the Nano/ALISA technology, and it contains:
1. the magnetic-particle that special aptamers is modified, about 1～1.5 μ m of its particle diameter, concentration is about 5mg ml
-1
2. special one of detection of Salmonella is anti-, optimum concentration 2 μ g ml
-1
3. two is anti--couplet of nm of gold-reporter groups, and optimum dilution degree is 1: 10;
4. confining liquid, its component is 10mM PBS (pH 7.4) solution that contains 1%BSA;
5. cleansing solution, its component is 10mM PBS (pH 7.4) solution that contains 0.05%Tween 20;
6. substrate, the corresponding substrate of reporter group in the nm of gold couplet, as 3,3 ', 5,5 '-tetramethyl benzidine (3,3 ', 5,5 '-tetramethylbenzidine, TMB) etc.;
7. positive control bacterium liquid and negative control bacterium liquid;
8. instructions.
Be used for the not special restriction of confining liquid of the present invention, cleansing solution, can adopt the corresponding confining liquid in this area, cleansing solution.In addition, confining liquid in the kit and cleansing solution can be to concentrate or dilution.
With respect to prior art, the invention provides a kind of novel immunological method and kit of fast detecting detection of Salmonella of with low cost, simple to operate, highly sensitive, high specificity; And proposed first to constitute sandwich structure being used for the novel immunological method of specific detection pathogen with aptamers, pathogen and nm of gold, for the detection of pathogen provides a kind of new thinking.Compare with traditional ELISA method, the present invention amplifies means with the Nano/ALISA technology as a kind of novel multilevel signal, be expected under the operation that only need be similar to elisa technique and appointed condition realization to various materials fast, detection delicately.This convenience helps this The Application of Technology and popularization, and the scene that is particularly suitable for the epidemic-stricken area pathogen is detected.
Description of drawings
Below in conjunction with accompanying drawing the present invention is further specified.
Fig. 1 (a) is the AFM figure of nm of gold; (b) be the TEM figure of nm of gold;
Fig. 2 is the uv-visible absorption spectroscopy figure of nm of gold;
Fig. 3 (a) is two anti-the righttest stable quantities; (b) be the righttest stable quantity of HRP;
The two anti-and HRP ratio Optimization result of Fig. 4 for modifying on the nm of gold couplet;
Fig. 5 is an anti-concentration Optimization result;
Fig. 6 is the work synoptic diagram based on the Salmonella typhimurtum method for quick of Nano/ALISA technology;
The result of Fig. 7 for the specificity of detection method of the present invention is studied;
The result of Fig. 8 for the sensitivity of detection method of the present invention is analyzed.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the embodiment that lifts not as to qualification of the present invention.
Below be instrument and equipment used in the part embodiment of the invention, other not marked experiment conditions are according to condition conventional or that advised with its manufacturer.
The used instrument of scanning that nano-Au solution carries out 400nm～800nm wavelength coverage is that (U-3010 Hitachi), measures with quartzy black matrix trace cuvette ultraviolet-visible pectrophotometer; Sample volume 100 μ l, room temperature, parameter setting: wave band scanning; 400nm～800nm, Scan Speed 1200nm/min, Sampling Interval 2nm; Slit Width 0.5nm, Lamp Change 339nm.It is that (Genios TECAN), measures room temperature with 96 orifice plates or 384 orifice plates to ELIASA that UV, visible light optical wavelength range internal absorbance detects used instrument.The particle diameter of nm of gold and shape characterize used instrument be transmission electron microscope (JEM-2011, JEOL) and atomic force microscope (NanoScope IIIa, Veeco).
The 15nm nm of gold adopts the preparation of trisodium citrate method: 0.01% the aqueous solution of chloraurate of 100ml is added the 250ml round-bottomed flask; After being heated to violent boiling; 1% trisodium citrate aqueous solution that adds 3.5ml under the vigorous stirring fast behind the continuation heated and stirred 15min, stops heating; Leave standstill the room temperature natural cooling after continuing to stir 30min.With 0.22 μ m membrane filtration, 4 ℃ of preservations.The nm of gold that is equipped with gained according to this legal system utilizes atomic force microscope and transmission electron microscope that its particle diameter and shape are characterized, and as shown in Figure 1, particle diameter ratio is than homogeneous, greatly between 15～20nm; Adopt ultraviolet-visible light spectrum spectrophotometer to characterize sample characteristic of correspondence absorption peak (as shown in Figure 2).The extinction coefficient epsilon that golden nanometer particle is corresponding
520Value gets 3.64 * 10
8L mol
-1Cm
-1, according to Lambert-Beer's law and the colloidal sol absorbance at the 520nm place, the concentration that can calculate aurosol is 2.2nM.
Used various solution composition is as shown in table 1.
Table 1 solution composition table
The aptamers of embodiment 1 magnetic-particle is modified
The specificity aptamers such as Salmonella typhimurtum that 5 ' end amination is modified are responsible for synthetic by Sangon Biotech (Shanghai) Co., Ltd. and are accomplished amido modified.Utilize the Maldi-Tof mass spectrophotometry to carry out quality control.Aptamers is all with aqua sterilisa dissolving, packing ,-20 ℃ of preservations.Before the use, 95 ℃ of sex change 5min, 4 ℃ of 10min pre-service.
Amino-magnetic particle binding reagents box is purchased the company in Bangs Laboratories, about 1～1.5 μ m of particle diameter, and solid content is 50mg ml
-1Press product description, get 100 μ l amino-magnetic particles to 1.5ml EP pipe; Add 0.8ml pyridine washing lotion (Pyridine Wash Buffer, PWB), mixing, magnetic separate, repeated washing 3 times; Add in 0.4ml 5% glutaraldehyde to the above-mentioned magnetic-particle mixing; Mixed at room temperature 3 hours; Magnetic separates, and abandons supernatant, repeated washing 4 times; Adding is with the amination aptamers 10 μ M 800 μ l of PWB dilution, behind the vortex oscillation mixing to 25 ℃ in constant-temperature shaking appearance, 16～24h; Magnetic separates, and it is resuspended to add 800 μ l PWB; Add behind 400 μ l quencher damping fluid (1M glycocoll, pH 8.0) the vortex oscillation mixings to 25 ℃ in constant-temperature shaking appearance, 30min; Add 800 μ l washing lotions (0.1mM EDTA, pH 7.4 for 10mM PBS, 0.1% BSA) vortex oscillation mixing, magnetic divides the abandonment supernatant, repeats 3 times; It is resuspended to add the 1ml washing lotion, and this moment, magnetic-particle concentration was about 5mg ml
-1, 2 ℃～8 ℃ preservations.
The preparation of embodiment 2 nm of gold couplets
1. protein to be marked is the mensuration of right stable quantity
The equal-volume protein solution (2 μ l) for preparing a series of variable concentrations adds respectively in 100 μ l (pH the transfers to 8.0-8.5) nm of gold, and rapidly mixing respectively adds 20 μ l, 10% NaCl solution then, shakes up, and surveys each pipe after leaving standstill 5min.According to the size of nanogold particle, measure the absorbance at 520nm place, be ordinate with the absorbance, the protein consumption is that horizontal ordinate is made a curve, gets the protein consumption that curve reaches the equilibrium point place at first and is the righttest stable quantity.
Shown in Fig. 3, the righttest stable quantity of two anti-(sheep anti-mouse igg is purchased the company in Fitzgerald) and horseradish peroxidase (HRP purchases the company in SIGMA) is respectively 25 μ g ml in the nano-Au solution of 15nm
-1With 35 μ g ml
-1
2. the optimization of two anti-and HRP ratio and nm of gold couplet extension rates
With 10mg ml
-1Sheep anti-mouse igg and HRP press different proportion (9: 1,4: 1,1: 1,1: 4,9: 1) and mix, and respectively get 5 μ l and add respectively in 1ml (pH transfers to 8.0～9.0) nm of gold, and 10min is hatched in 37 ℃ of vibrations, 4 ℃ of hold over night.Add 5% PEG6000 to final concentration 0.5%, mixing was placed 1 hour for 4 ℃.The centrifugal 20min of 12000rpm removes supernatant, adds store buffer liquid 1ml, and is resuspended.Repeat this centrifuge washing step, 3 times.With 200 μ l store buffer liquid mixings, place 4 ℃ of preservations at last.
With 2 μ g ml
-1Special one anti-(dilution of 0.05M carbonate buffer solution) 20 μ l of Salmonella typhimurtum are sub-packed in elisa plate 384 micropores, and 4 ℃ encapsulate and spend the night; Discard solution in the hole, wash plate 3 times with carbonate buffer solution; The nm of gold couplet of above-mentioned different proportion sheep anti-mouse igg and HRP preparation is diluted (1,2.5,5,10,20 times) by a certain percentage, add in the above-mentioned corresponding micropore, hatch 1h for 37 ℃, do blank simultaneously; Discard solution in the hole, wash plate 3 times with cleansing solution; Add TMB 20 μ l, room temperature 10min; Add stop buffer 20 μ l, room temperature 5min; Under the 450nm wavelength, read the A value.Shown in Figure 4, two anti-and HRP optimal proportionss are 1: 1, and promptly two anti-molal quantitys with HRP are respectively 76 times and 258 times of nm of gold; And the optimum diluting multiple of nm of gold couplet is 10 times.
The optimization of embodiment 3 one anti-concentration
1. draw 2 μ l aptamers modified magnetic particles, magnetic divides the abandonment supernatant;
2. add confining liquid 1%BSA-PBS 20 μ l, 37 ℃, 650rpm, 0.5h, concussion mixing;
3. add the bacterium liquid 200 μ l with sterilization PBS dilution, 650rpm is hatched 1h for 37 ℃, discards reactant liquor, with 200 μ l cleansing solutions washing 3 times;
4. add variable concentrations one anti-(0.1,0.5,2,10,50 μ g ml with the confining liquid dilution
-1) 10 μ l, 650rpm is hatched 1h for 37 ℃;
5. discard reactant liquor, with cleansing solution washing 3 times;
6. add the nm of gold couplet 10 μ l of 10 times of dilutions, hatch 1h for 37 ℃;
7. discard reactant liquor, with cleansing solution washing 5 times;
8. add 100 μ l TMB, room temperature vibration 10 minutes is read the 595nm light absorption value with ELIASA.
As shown in Figure 5, an anti-concentration is 2 μ g ml
-1The time, signal to noise ratio (S/N ratio) (signal-to-noise ratio, S/N) the highest, be 2.68 ± 0.24, i.e. 2 μ g ml
-1Be the righttest one anti-concentration.
The synoptic diagram of fast detecting Salmonella typhimurtum of the present invention such as Fig. 6, concrete steps comprise:
(1) draw the special aptamers modified magnetic of 2 μ l Salmonella typhimurtums particle, magnetic divides the abandonment supernatant;
(2) add confining liquid 1%BSA-PBS 20 μ l, 37 ℃, 650rpm, 0.5h shakes mixing;
(3) add the bacterium liquid 200 μ l that dilute with sterilization PBS, 650rpm is hatched 1h for 37 ℃, discards reactant liquor, with 200 μ l cleansing solutions washing 3 times;
(4) add the 2 μ g ml that dilute with confining liquid
-1Variable concentrations one anti-10 μ l, 650rpm is hatched 1h for 37 ℃;
(5) all the other steps are with 5～7 of embodiment 3.
1. cultivation of bacterium and counting:
The single bacterium colony of picking is to 5ml LB fluid nutrient medium, and 36 ± 1 ℃ of 200rpm/min cultivate 16h～18h, makes it reach the required concentration requirement of experiment.Use 5ml PBS water fully resuspended again after washing 2 times with sterilization PBS then.The thalline solution that washing is good is diluted to the even liquid of 8 10 times of serial dilution samples successively, is respectively charged into 8 sterile vials, from 10
1To 10
8, every bottle of 1ml, label 1～8 respectively.Traditional plate count method is used in cell count, and each dilutability is got 100 μ l respectively, is uniformly coated on three plain agar planar surfaces, behind 36 ± 1 ℃ of cultivation 24h, and the clump count of naked eyes counting readable region on flat board.Choose clump count between 30CFU～300CFU, do not have the plate count total plate count spread colony growth, calculate the bacterium colony average on three flat boards of same dilutability, and calculate by following formula:
Average clump count * extension rate * 10 that concentration (CFU/ml)=same dilutability repeats for three times
Getting 50 μ l sterilization PBS simultaneously respectively is uniformly coated on three plain agar flat boards and does blank.After the culture counting was accomplished, the bottle that viable bacteria is housed was preserved in 4 ℃ of refrigerators.
2. specificity experiment
Select gram-negative bacteria (typhoid fever sramana CMCC 50071, S.typhi; Paratyphosus A bacillus CMCC50001, S.paratyphi A; Song Nei Shi shigella dysenteriae, S.sonnei; Shigella flexneri CMCC 51571, S.flexneri; Escherichia coli O 157 CICC 21530, E.coli O157; Escherichia coli O111 CMCC 44151, E.coli O111; Escherichia coli ATCC 25922, E.coli ATCC 25922) and gram positive bacteria (staphylococcus aureus ATCC25923 S.aureus) studies specificity of the present invention.After the incubated overnight, (CMCC 50115, and bacterial concentration S.typhimurium) adjusts to 10 with above-mentioned bacterium and Salmonella typhimurtum respectively
8CFU ml
-1, detect as stated above, read the 595nm light absorption value with ELIASA, and the ratio of calculating and Salmonella typhimurtum testing result.As shown in Figure 7; Use the kit of the special detection of Salmonella typhimurtum of the present invention's design that Salmonella typhi, paratyphosus A bacillus, Song Nei Shi shigella dysenteriae, Shigella flexneri etc. are detected; Only Salmonella typhimurtum is positive, and all the other bacteriums are all negative, and the A value is all less than A
S.typhimurium11%, cross reaction or nonspecific reaction can not take place all.
3. sensitivity experiment
Use kit of the present invention to detect the Salmonella typhimurtum (PBS dilution) of variable concentrations, the result is as shown in Figure 8.Along with the increase of Salmonella typhimurtum concentration, the A value increases gradually, detects and is limited to 10
3CFU ml
-1(S/N＞3).
Claims (9)
1. the detection of Salmonella method for quick based on the Nano/ALISA technology is characterized in that, may further comprise the steps successively:
(1) the special aptamers of detection of Salmonella is modified the magnetic-particle surface;
(2) adding sample reacts;
(3) special one of the adding detection of Salmonella anti-the reaction;
(4) couplet that adds two anti--nm of gold-reporter groups is reacted;
(5) add specific substrate and react, or directly write down the light signal of this moment.
2. the detection of Salmonella method for quick based on the Nano/ALISA technology according to claim 1 is characterized in that, has proposed first to constitute sandwich structure to be used for the novel immunological method of specific detection pathogen with aptamers, pathogen and nm of gold.
3. the detection of Salmonella method for quick based on the Nano/ALISA technology according to claim 1 is characterized in that the particle diameter of the said nm of gold of step (4) is 15～20nm.
4. the detection of Salmonella method for quick based on the Nano/ALISA technology according to claim 1 is characterized in that, the said reporter group of step (4) have the enzyme of color or fluorescence signal for the ability catalytic substrate generates, or has the fluorescin of autoluminescence performance.
5. the detection of Salmonella method for quick based on the Nano/ALISA technology according to claim 1 is characterized in that, the preparation method of the couplet of step (4) said two anti--nm of gold-reporter groups comprises the following steps:
(1) anti-mix by a certain percentage with reporter group two, join in the nano-Au solution, regulate the pH value, 10min is hatched in 37 ℃ of vibrations, 4 ℃ of hold over night;
(2) add the surperficial vacant binding site of confining liquid sealing nm of gold;
(3) centrifuge washing is removed not bound substances;
(4) add the storage liquid mixing, place 4 ℃ of preservations.
6. preparation method according to claim 5 is characterized in that step (1) described two anti-molal quantitys with reporter group are respectively 50～100 times and 200～400 times of nm of gold, and the pH value is 8.0～9.0.
7. preparation method according to claim 5 is characterized in that the described centrifugal condition of step (3) is the centrifugal 20min of 12000rpm.
8. preparation method according to claim 5 is characterized in that step (3) and (4) described cleansing solution and storage liquid consist of 0.5% bovine serum albumin(BSA), 2.5% sucrose, 0.1%PEG, 10mM PBS (pH7.4).
9. the detection of Salmonella quick detection kit based on the Nano/ALISA technology is characterized in that, contains in the kit:
(1) magnetic-particle of special aptamers modification, about 1～1.5 μ m of its particle diameter, concentration is about 5mg ml
-1
(2) special one of detection of Salmonella is anti-, optimum concentration 2 μ g ml
-1
The couplet of (3) two anti--nm of gold-reporter groups, optimum dilution degree is 1: 10;
(4) confining liquid, its component are 10mM PBS (pH 7.4) solution that contains 1%BSA;
(5) cleansing solution, its component are 10mM PBS (pH 7.4) solution that contains 0.05% Tween 20;
(6) substrate, the corresponding substrate of reporter group in the nm of gold couplet, as 3,3 ', 5,5 '-tetramethyl benzidine (3,3 ', 5,5 '-tetramethylbenzidine, TMB) etc.;
(7) positive control bacterium liquid and negative control bacterium liquid;
(8) instructions.
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Cited By (9)
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| CN103014163A (en) * | 2012-12-19 | 2013-04-03 | 江南大学 | Visual detection method for mouse typhus salmonella based on aptamer recognition |
| CN103913446A (en) * | 2014-02-28 | 2014-07-09 | 江南大学 | Detection method for food-borne pathogenic bacteria by using sensor based on dye AccuBlue label-free aptamer |
| CN103983783A (en) * | 2014-06-10 | 2014-08-13 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for detecting pathogenic bacteria and application of kit |
| WO2015039423A1 (en) * | 2013-09-18 | 2015-03-26 | 深圳迈瑞生物医疗电子股份有限公司 | Biomarker preservative solution, reagent and method |
| CN104928257A (en) * | 2015-05-26 | 2015-09-23 | 扬州大学 | Hybridoma cell strain secreting salmonella SpiC protein monoclonal antibody, monoclonal antibody thereof, and application of monoclonal antibody |
| CN107084976A (en) * | 2017-04-18 | 2017-08-22 | 江南大学 | A kind of method recognized based on aptamers with Mimetic Peroxidase Visual retrieval salmonella |
| CN107607506A (en) * | 2017-09-05 | 2018-01-19 | 中山大学 | A kind of quick detection platform based on the micro-nano probe of magnetic coupling and micro-fluidic chip |
| CN108535482A (en) * | 2018-04-11 | 2018-09-14 | 上海萨迦生物科技有限公司 | A kind of antibody chip kit for tumour early screening and diagnosis |
| CN111796092A (en) * | 2020-08-17 | 2020-10-20 | 青岛农业大学 | pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014163A (en) * | 2012-12-19 | 2013-04-03 | 江南大学 | Visual detection method for mouse typhus salmonella based on aptamer recognition |
| WO2015039423A1 (en) * | 2013-09-18 | 2015-03-26 | 深圳迈瑞生物医疗电子股份有限公司 | Biomarker preservative solution, reagent and method |
| CN103913446A (en) * | 2014-02-28 | 2014-07-09 | 江南大学 | Detection method for food-borne pathogenic bacteria by using sensor based on dye AccuBlue label-free aptamer |
| CN103983783A (en) * | 2014-06-10 | 2014-08-13 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for detecting pathogenic bacteria and application of kit |
| CN103983783B (en) * | 2014-06-10 | 2016-03-23 | 江苏出入境检验检疫局动植物与食品检测中心 | A kind of kit and application thereof detecting pathogenic bacteria |
| CN104928257A (en) * | 2015-05-26 | 2015-09-23 | 扬州大学 | Hybridoma cell strain secreting salmonella SpiC protein monoclonal antibody, monoclonal antibody thereof, and application of monoclonal antibody |
| CN107084976A (en) * | 2017-04-18 | 2017-08-22 | 江南大学 | A kind of method recognized based on aptamers with Mimetic Peroxidase Visual retrieval salmonella |
| CN107084976B (en) * | 2017-04-18 | 2019-07-30 | 江南大学 | A kind of method of aptamers Mimetic Peroxidase Visual retrieval salmonella |
| CN107607506A (en) * | 2017-09-05 | 2018-01-19 | 中山大学 | A kind of quick detection platform based on the micro-nano probe of magnetic coupling and micro-fluidic chip |
| CN108535482A (en) * | 2018-04-11 | 2018-09-14 | 上海萨迦生物科技有限公司 | A kind of antibody chip kit for tumour early screening and diagnosis |
| CN111796092A (en) * | 2020-08-17 | 2020-10-20 | 青岛农业大学 | pH response-based heterochromatic nanoparticles, pathogen detection kit containing nanoparticles and detection method |
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Application publication date: 20120711 |