CN107505298B - Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe - Google Patents
Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe Download PDFInfo
- Publication number
- CN107505298B CN107505298B CN201710705105.0A CN201710705105A CN107505298B CN 107505298 B CN107505298 B CN 107505298B CN 201710705105 A CN201710705105 A CN 201710705105A CN 107505298 B CN107505298 B CN 107505298B
- Authority
- CN
- China
- Prior art keywords
- cyromazine
- detection
- milk
- solution
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- LVQDKIWDGQRHTE-UHFFFAOYSA-N cyromazine Chemical compound NC1=NC(N)=NC(NC2CC2)=N1 LVQDKIWDGQRHTE-UHFFFAOYSA-N 0.000 title claims abstract description 85
- 239000005891 Cyromazine Substances 0.000 title claims abstract description 83
- 229950000775 cyromazine Drugs 0.000 title claims abstract description 83
- 235000013336 milk Nutrition 0.000 title claims abstract description 37
- 239000008267 milk Substances 0.000 title claims abstract description 37
- 210000004080 milk Anatomy 0.000 title claims abstract description 37
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 61
- 238000010791 quenching Methods 0.000 claims description 23
- 230000000171 quenching effect Effects 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 19
- 239000000523 sample Substances 0.000 claims description 18
- 230000005284 excitation Effects 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000012086 standard solution Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 5
- 239000008351 acetate buffer Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 abstract description 10
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 abstract description 7
- 108091081406 G-quadruplex Proteins 0.000 abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 abstract description 6
- 108020004707 nucleic acids Proteins 0.000 abstract description 5
- 102000039446 nucleic acids Human genes 0.000 abstract description 5
- 229940113082 thymine Drugs 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 description 8
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 description 7
- 239000002207 metabolite Substances 0.000 description 7
- 229920000877 Melamine resin Polymers 0.000 description 6
- 238000002983 circular dichroism Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000004100 Oxytetracycline Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- MXWJVTOOROXGIU-UHFFFAOYSA-N atrazine Chemical compound CCNC1=NC(Cl)=NC(NC(C)C)=N1 MXWJVTOOROXGIU-UHFFFAOYSA-N 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 2
- 239000010871 livestock manure Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 description 2
- 229960000625 oxytetracycline Drugs 0.000 description 2
- 235000019366 oxytetracycline Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- HWRKQCBVHKEFKX-UHFFFAOYSA-N 2-n-cyclopropyl-1h-triazine-2,4,6-triamine Chemical compound N1C(N)=CC(N)=NN1NC1CC1 HWRKQCBVHKEFKX-UHFFFAOYSA-N 0.000 description 1
- IBSREHMXUMOFBB-JFUDTMANSA-N 5u8924t11h Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C(C)C)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 IBSREHMXUMOFBB-JFUDTMANSA-N 0.000 description 1
- 239000005660 Abamectin Substances 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 239000005500 Clopyralid Substances 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 208000006568 Urinary Bladder Calculi Diseases 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229950008167 abamectin Drugs 0.000 description 1
- QXAITBQSYVNQDR-ZIOPAAQOSA-N amitraz Chemical compound C=1C=C(C)C=C(C)C=1/N=C/N(C)\C=N\C1=CC=C(C)C=C1C QXAITBQSYVNQDR-ZIOPAAQOSA-N 0.000 description 1
- 229960002587 amitraz Drugs 0.000 description 1
- YSKUZVBSHIWEFK-UHFFFAOYSA-N ammelide Chemical compound NC1=NC(O)=NC(O)=N1 YSKUZVBSHIWEFK-UHFFFAOYSA-N 0.000 description 1
- MASBWURJQFFLOO-UHFFFAOYSA-N ammeline Chemical compound NC1=NC(N)=NC(O)=N1 MASBWURJQFFLOO-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229940097572 chloromycetin Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- HUBANNPOLNYSAD-UHFFFAOYSA-N clopyralid Chemical compound OC(=O)C1=NC(Cl)=CC=C1Cl HUBANNPOLNYSAD-UHFFFAOYSA-N 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000020335 dealkylation Effects 0.000 description 1
- 238000006900 dealkylation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229960001614 levamisole Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 150000007974 melamines Chemical class 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- AAEVYOVXGOFMJO-UHFFFAOYSA-N prometryn Chemical compound CSC1=NC(NC(C)C)=NC(NC(C)C)=N1 AAEVYOVXGOFMJO-UHFFFAOYSA-N 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- ODCWYMIRDDJXKW-UHFFFAOYSA-N simazine Chemical compound CCNC1=NC(Cl)=NC(NCC)=N1 ODCWYMIRDDJXKW-UHFFFAOYSA-N 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 1
- 229960003053 thiamphenicol Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 150000003918 triazines Chemical class 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe. When the detection system does not contain cyromazine, fluorescein FAM modified on the aptamer releases fluorescence at 520 nm; when cyromazine is added into a system, cyromazine is combined with thymine on functional nucleic acid to promote the functional nucleic acid to form a G-quadruplex structure, so that the fluorescence release at 520nm is inhibited, and the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal based on that the released fluorescence signal is in inverse proportion to the concentration of cyromazine. The method can be used for detecting the cyromazine in the milk, and has the advantages of high detection sensitivity, low detection limit of 0.68ppb, good selectivity, simple and convenient operation, simple pretreatment and no need of large-scale instruments.
Description
Technical Field
The invention belongs to the technical field of cyromazine detection, and particularly relates to a method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe.
Background
Cyromazine (Cyr) is 2-cyclopropylamino-4, 6-diaminotriazine, and belongs to triazine or triazine compounds such as atrazine, simazine, atrazine and prometryn, and is a high-efficiency insect growth inhibitor and parasite killing pesticide, and is used as a feed additive in the process of livestock and poultry breeding. Studies have shown that cyromazine enters animals and is excreted in the animal body mostly as bulk drug or metabolite by milk or manure, and is subsequently exposed to soil and water environment via livestock manure. The cyromazine in soil or water environment enters the food chain again through environmental return, and potential hidden dangers are caused to organisms with different nutritional levels and the health of human bodies. Cyromazine is metabolized to Melamine (Mel) by dealkylation in animals and plants, and the main metabolites of Melamine are Cyanuric Acid (CA), Ammelide (Amd), and Ammeline (Amn). Long-term involvement of melamine can cause bladder stones, and the incidence rate of bladder cancer is obviously improved.
The rapid detection of the cyromazine and the metabolites thereof in the milk can effectively prevent toxic and harmful substances from entering a human body. Cyromazine and its metabolite melamine are a group of compounds with small molecular weight and strong polarity, and it is very difficult to extract cyromazine and its related metabolites from actual samples. The united states EPA has strictly regulated that cyromazine must be a feed additive and can only be used in stable houses and feed troughs, but still flies are killed by spraying for use in breeding places, compost, garbage, etc. After the cyromazine is orally taken by animals, most of the cyromazine is excreted in the form of raw medicine or metabolite melamine through urine and feces of the animals, and little residue is left in the animals. The U.S. EPA and PRC set the highest residue standard for cyromazine in a range of animal and vegetable foods, with milk at 0.05 mg/kg.
According to the limited standard requirements of the cyromazine and the metabolite melamine thereof in food, a capacity analysis method is commonly used for detecting the residue of the cyromazine and the metabolite thereof at present; chromatographic methods, including high performance liquid chromatography; immunochemical assays; optical analysis, and the like. The residue of the cyromazine in the animal food is determined by adopting ultra performance liquid chromatography-tandem mass spectrometry in GB 29704-2013. With the fact that the residue detection of the cyromazine veterinary drug is listed in the essential items of raw milk purchase and dairy product delivery, due to the limitation of the detection method and conditions, the existing detection method is objectively required to be improved in the face of huge detection quantity, which causes huge cost and heavy workload and is difficult to meet the requirement of on-site rapid detection, and a high-throughput, rapid and high-sensitivity cyromazine detection method is developed for daily monitoring.
The aptamer is a nucleic acid fragment which has high specificity and high tuberculous property to a target substance and is screened from a large oligonucleotide library by an exponential enrichment ligand system in vitro evolution (SELEX) technology. However, compared with protein antibodies and biological enzymes, aptamers have higher affinity, stability and specificity, and are easy to label to design sensors, and the sensors are already used for detecting nucleic acids, proteins, inorganic metal ions, virus particles and cells. Although it has been reported that thymine can bind to melamine, which is a metabolite of cyromazine, through hydrogen bonds, aptamers that can specifically bind to cyromazine to form a G-quadruplex have not been screened or synthesized, and the use of a fluorescence spectrophotometer for detection based on this has not been reported.
Disclosure of Invention
The invention aims to provide a detection method of cyromazine in milk, which has high sensitivity, good selectivity and low cost by utilizing the phenomenon that cyromazine with different concentrations can quench the fluorescence intensity of fluorescein in detection liquid to different degrees.
In order to achieve the aim, the invention provides a method for detecting the cyromazine in the milk by utilizing a fluorescein FAM modified aptamer detection solution capable of forming a G-quadruplex. The fluorescein FAM is modified at the 5' end of the aptamer to form a stable detection solution, and when the wavelength of excitation light is 480nm, stable fluorescence is released at 520 nm.
The principle of the invention is as follows: according to the structural characteristics of the cyromazine, the high-specificity aptamer of the cyromazine which mainly takes thymine and is rich in guanine is designed and synthesized by combining the property that the cyromazine can be combined with thymine through a hydrogen bond, and fluorescein FAM is modified at the 5' end. The existence of cyromazine can change the spatial structure of an aptamer to form a stable G-quadruplex structure. Due to the change in the steric structure of the aptamer, the distance between the above-attached fluorophore and cyromazine becomes small, thereby quenching fluorescence. By the relationship between the concentration of cyromazine and the fluorescence quenching rate, i.e. the quenching rate F0The value of/F is linear with the concentration of cyromazine in a certain range, so that the fluorescence quenching rate F is analyzed0The quantitative detection of the cyromazine in the milk can be realized by the change of the/F.
The technical scheme of the invention is specifically introduced as follows.
The invention provides a method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe, which comprises the following steps:
(1) mixing a plurality of detection liquids with the same concentration and a plurality of pretreated milk samples added with the standard solutions of the cyromazine with different concentrations for 2min at the temperature of 24-26 ℃ to obtain a standard solution with the cyromazine content maintained between 0ppb and 10 ppb; wherein: the detection solution is obtained by diluting FAM modified aptamer with Tris-acetate buffer solution, wherein the FAM modified aptamer is 5' -FAM-GGTTGGTTGGTTGGTTTT-3;
(2) placing the prepared standard solution in a cuvette, setting the excitation wavelength to be 480nm, and measuring the fluorescence intensity of the standard solution at 520nm by using a fluorescence spectrophotometer; the fluorescence intensity measured when cyromazine was present in the detection system was designated as F, and the fluorescence intensity measured when cyromazine was not present was designated as F0The quenching rate (F) was calculated0-F)/F0；
(3) Cyromazine at different concentrations and corresponding quenching rates (F)0-F)/F0Drawing, and drawing a standard curve;
(4) pretreating a milk sample to be detected, mixing the pretreated milk sample with the detection solution with the same concentration as that in the step (1), reacting for 1-3 min after fully and uniformly mixing, testing the fluorescence intensity of the mixed solution at 520nm by using a fluorescence spectrophotometer at an excitation wavelength of 480nm, and calculating the quenching rate (F)0-F)/F0；
(5) According to the quenching rate (F) of the milk sample to be detected0-F)/F0And checking a standard curve to obtain the cyromazine content in the milk sample to be detected.
In the invention, the concentration of the detection solution is 20-30 nmol/L.
In the invention, the pretreatment of the milk sample comprises the following steps: the pretreatment of the milk sample comprises the following steps: mixing 4-6 mL of 1 wt% acetic acid and 1.0mL of milk sample, and standing for 5 minutes; then centrifuging at 8000-12000 r/min for 8-12 minutes, taking the supernatant, and adjusting the pH to 8.0 by using 1.5-2.5 mol/L NaOH.
Compared with the prior art, the invention has the beneficial effects that: the detection method provided by the method has high sensitivity and good selectivity, the minimum detection limit is 0.68ppb, the operation is simple and convenient, the pretreatment is simple, large-scale instruments are not needed, and the method can be used for detecting the cyromazine in the milk.
Drawings
FIG. 1 is a schematic diagram of the principle of rapid detection of cyromazine based on a G-quadruplet aptamer fluorescent probe.
FIG. 2 is a circular dichroism chart of the detection system.
FIG. 3 is a graphical representation of the effect of temperature on the detection system.
FIG. 4 shows reaction time and K+Graphical representation of the effect on the detection system.
FIG. 5 shows the results of the system selectivity experiment.
FIG. 6 shows the cyromazine concentration and fluorescence quenching rate (F)0-F)/F0The relationship (2) of (c).
Detailed Description
The technical solution of the present invention will be described in detail with reference to the accompanying drawings and embodiments.
FIG. 1 is a schematic diagram of the principle of rapid detection of cyromazine based on a G-quadruplet aptamer fluorescent probe. When the detection system does not contain cyromazine, fluorescein FAM modified on the aptamer releases fluorescence at 520 nm; when cyromazine is added into a system, cyromazine is combined with thymine on functional nucleic acid to promote the functional nucleic acid to form a G-quadruplex structure, so that the fluorescence release at 520nm is inhibited, and the quantitative detection of cyromazine in milk can be realized by measuring the fluorescence signal based on that the released fluorescence signal is in inverse proportion to the concentration of cyromazine.
Example 1
(1) And (3) system circular dichroism detection: preparing two centrifuge tubes, respectively adding aptamer and aptamer-mixed cyromazine, and scanning by circular dichroism at a wavelength of 220-320 nm to prove the formation of a G-quadruplex structure. FIG. 2 is a circular dichroism chart of the detection system.
(2) Influence of temperature on the detection system: a cyromazine test solution mixed system with cyromazine final concentrations of 0ppb,6ppb,15ppb,40ppb,100ppb and 200ppb was prepared, and the fluorescence intensity at 520nm was measured with a fluorescence spectrophotometer under the conditions of 25 ℃,35 ℃,45 ℃ and 55 ℃ after setting the excitation wavelength at 480 nm. The fluorescence intensity measured when cyromazine was present in the detection system was designated as F, and the fluorescence intensity measured when cyromazine was not present was designated as F0Calculating F0and/F. FIG. 3 is a graphical representation of the effect of temperature on the detection system, as shown by the temperature at 25 ℃ being optimal.
(3)K+Influence on the detection System: respectively preparing 0,10, 50 and 100ppb K+And mixing the solution with 25nM aptamer, and setting the excitation wavelength to 480nM and measuring the fluorescence intensity of the solution at 520nM by using a fluorescence spectrophotometer in the process of reacting with the detection solution for 0-360 seconds. Detecting the presence of K in the System+The fluorescence intensity measured is denoted F, K is absent+The measured fluorescence intensity was recorded as F0The quenching rate (F) was calculated0-F)/F0. Shown in FIG. 4, K+Influence diagram on the detection System, K+The increase in concentration does not affect the fluorescence quenching rate of the system.
(4) Influence of reaction time on the detection system: 0ppb,10 ppb, 50ppb and 100ppb cyromazine solutions are prepared respectively, and in the process of reacting with a detection solution for 0-360 seconds, the fluorescence intensity of the detection solution at 520nm is measured by a fluorescence spectrophotometer with an excitation wavelength of 480 nm. The fluorescence intensity measured when cyromazine was present in the detection system was designated as F, and the fluorescence intensity measured when cyromazine was not present was designated as F0The quenching rate (F) was calculated0-F)/F0. FIG. 4 is a graph showing the reaction time and the influence on the detection system, and it is understood from the graph that the optimum reaction time of the system is 2 minutes or less.
(5) And (3) selective test of a detection system: respectively adding 15ppb cyromazine and 50ppb of common residual pollutants oxytetracycline, thiamphenicol, chloromycetin, amitraz, clopyralid, abamectin, levamisole and oxytetracycline in milk samples, setting the excitation wavelength to 480nm, and measuring the fluorescence intensity of the samples at 520nm by using a fluorescence spectrophotometer. The fluorescence intensity measured in the presence of the veterinary drug in the detection system is denoted as F, and the fluorescence intensity measured in the absence is denoted as F0The quenching rate (F) was calculated0-F)/F0. FIG. 5 is a graph showing the results of selectivity measurement of the detection system, and it can be seen that the detection system has better selectivity.
Example 2
(1) Preparing a detection solution: and connecting fluorescein FAM to the 5 ' end of ssDNA with the sequence of GGTTGGTTGGTTGGTTTT to obtain the final cyromazine aptamer 5 ' -FAM-GGTTGGTTGGTTGGTTTT-3 '. The aptamer content was diluted to a concentration of 25nM with Tris-acetate buffer (10mM, pH 8.0) to obtain a test solution.
(2) Pretreatment of a milk sample: 5mL of 1% acetic acid and 1.0mL of a milk sample were added to a 10mL centrifuge tube, and the mixture was allowed to stand for 5 minutes. Then, the mixture was centrifuged at 10000r for 10 minutes, and after taking the supernatant, the pH was adjusted to 8.0 with 2.0mol/L NaOH.
(3) Preparing a detection system with known cyromazine concentration: and (3) taking 10 centrifuge tubes containing detection liquid, respectively adding the pretreated milk samples added with the standard cyromazine liquid with different concentrations to ensure that the cyromazine content in the whole detection system is maintained at 0-10ppb, and reacting at 25 ℃ for 2min and then determining.
(4) The prepared standard solution is placed in a cuvette, and the fluorescence intensity at 520nm is measured by a fluorescence spectrophotometer after the excitation wavelength is set to 480 nm. The fluorescence intensity measured when cyromazine was present in the detection system was designated as F, and the fluorescence intensity measured when cyromazine was not present was designated as F0The quenching rate (F) was calculated0-F)/F0。
(5) Cyromazine at different concentrations and corresponding quenching rates (F)0-F)/F0Drawing and drawing a standard curve. FIG. 6 shows the cyromazine concentration and fluorescence quenching rate (F)0-F)/F0Wherein the cyromazine concentration is 0,1,2,3,4,5,6,7,8,9,10,15,20,40,60,80,100and 200ppb, respectively, and the Tcy2 concentration is 25 nM.
(6) Preparing a sample to-be-tested system: after pretreatment, mixing with detection solution, reacting for 2min after fully mixing, and determining the quenching rate (F) according to the step (4)0-F)/F0。
(7) Quenching rate obtained according to sample (F)0-F)/F0And checking a standard curve to obtain the cyromazine content in the sample.
(8) And (3) verification: the method of the present invention is used to determine four portions of milk containing cyromazine with the concentration of 0.1, 0.2, 0.5 and 1.0ppm respectively, and the obtained recovery rates are 120%, 95%, 106% and 106% respectively, thus proving the reliability of the method.
(9) The method of the invention determines the minimum detection limit of the cyromazine in the milk to be 0.68 ppb.
Claims (3)
1. A method for detecting cyromazine in milk based on a G-quadruplet aptamer fluorescent probe is characterized by comprising the following specific steps:
(1) mixing a plurality of detection liquids with the same concentration and a plurality of pretreated milk samples added with the standard solutions of the cyromazine with different concentrations for 2min at the temperature of 24-26 ℃ to obtain a standard solution with the cyromazine content maintained between 0ppb and 10 ppb; wherein: the detection solution is obtained by diluting FAM modified aptamer with Tris-acetate buffer solution, wherein the FAM modified aptamer is 5' -FAM-GGTTGGTTGGTTGGTTTT-3;
(2) placing the prepared standard solution in a cuvette, setting the excitation wavelength to be 480nm, and measuring the fluorescence intensity of the standard solution at 520nm by using a fluorescence spectrophotometer; the fluorescence intensity measured when cyromazine was present in the detection system was designated as F, and the fluorescence intensity measured when cyromazine was not present was designated as F0The quenching rate (F) was calculated0-F)/F0；
(3) Cyromazine at different concentrations and corresponding quenching rates (F)0-F)/F0Drawing, and drawing a standard curve;
(4) pretreating a milk sample to be detected, mixing the pretreated milk sample with the detection solution with the same concentration as that in the step (1), reacting for 1-3 min after fully and uniformly mixing, testing the fluorescence intensity of the mixed solution at 520nm by using a fluorescence spectrophotometer at an excitation wavelength of 480nm, and calculating the quenching rate (F)0-F)/F0；
(5) According to the quenching rate (F) of the milk sample to be detected0-F)/F0And checking a standard curve to obtain the cyromazine content in the milk sample to be detected.
2. The method according to claim 1, wherein the concentration of the detection solution is 20 to 30 nmol/L.
3. The method of claim 1, wherein the step of pre-treating the milk sample comprises: mixing 4-6 mL of 1 wt% acetic acid and 1.0mL of milk sample, and standing for 5 minutes; then centrifuging at 8000-12000 r/min for 8-12 minutes, taking the supernatant, and adjusting the pH to 8.0 by using 1.5-2.5 mol/L NaOH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710705105.0A CN107505298B (en) | 2017-08-17 | 2017-08-17 | Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710705105.0A CN107505298B (en) | 2017-08-17 | 2017-08-17 | Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107505298A CN107505298A (en) | 2017-12-22 |
| CN107505298B true CN107505298B (en) | 2020-05-12 |
Family
ID=60691091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710705105.0A Expired - Fee Related CN107505298B (en) | 2017-08-17 | 2017-08-17 | Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107505298B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109115705A (en) * | 2018-08-31 | 2019-01-01 | 上海应用技术大学 | A method of utilizing cyromazine in aptamer and PDDA assemble nanometer detection of particles milk |
| CN109444098A (en) * | 2018-11-05 | 2019-03-08 | 青岛科技大学 | A kind of biological sensor and its preparation method and application based on cyclic amplification technology and carboxyl carbon quantum dot |
| CN109444101B (en) * | 2018-12-13 | 2021-03-02 | 四川大学 | Proportional aptamer fluorescent probe and method for detecting ochratoxin A by using same |
| CN110567936B (en) * | 2019-09-05 | 2021-07-20 | 上海应用技术大学 | Method for detecting cyromazine in milk based on nucleic acid aptamer |
| CN113125399A (en) * | 2021-04-14 | 2021-07-16 | 四川大学 | Lead ion detection method based on G-quadruplex proportion fluorescence |
| CN114280286A (en) * | 2021-12-16 | 2022-04-05 | 上海交通大学云南（大理）研究院 | Method for detecting metamitron by adopting SYBR GREEN I fluorescence method based on aptamer |
| CN114397282B (en) * | 2021-12-24 | 2023-11-17 | 江苏鑫蓝鑫生物科技有限公司 | Method for detecting chloramphenicol by using nucleic acid aptamer and G-quadruplex label-free fluorescence analysis method |
| CN115044587B (en) * | 2022-05-23 | 2023-04-07 | 江南大学 | Aptamer capable of specifically recognizing levamisole, and dual-mode nanoprobe and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN104897585A (en) * | 2015-05-06 | 2015-09-09 | 同济大学 | Preparation method of aptamer colorimetric sensor for MC-LR fast detection |
| CN106680255A (en) * | 2017-01-04 | 2017-05-17 | 吉林大学 | Detection of melamine based on G-quadruplet aptamer fluorescent probe |
| CN107064263A (en) * | 2017-05-09 | 2017-08-18 | 同济大学 | Aptamer photoelectric sensor for detecting Atrazine and preparation method thereof |
| CN107153041A (en) * | 2017-05-11 | 2017-09-12 | 同济大学 | The preparation and application for the aptamer colorimetric sensor that Polychlorinated biphenyls 77 is detected |
-
2017
- 2017-08-17 CN CN201710705105.0A patent/CN107505298B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN104897585A (en) * | 2015-05-06 | 2015-09-09 | 同济大学 | Preparation method of aptamer colorimetric sensor for MC-LR fast detection |
| CN106680255A (en) * | 2017-01-04 | 2017-05-17 | 吉林大学 | Detection of melamine based on G-quadruplet aptamer fluorescent probe |
| CN107064263A (en) * | 2017-05-09 | 2017-08-18 | 同济大学 | Aptamer photoelectric sensor for detecting Atrazine and preparation method thereof |
| CN107153041A (en) * | 2017-05-11 | 2017-09-12 | 同济大学 | The preparation and application for the aptamer colorimetric sensor that Polychlorinated biphenyls 77 is detected |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107505298A (en) | 2017-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107505298B (en) | Method for detecting cyromazine in milk based on G-quadruplet aptamer fluorescent probe | |
| Kantiani et al. | Analytical methodologies for the detection of β-lactam antibiotics in milk and feed samples | |
| Bickman et al. | An innovative portable biosensor system for the rapid detection of freshwater cyanobacterial algal bloom toxins | |
| Msagati et al. | Evaluation of methods for the isolation, detection and quantification of cyanobacterial hepatotoxins | |
| Crews | Analysis of ergot alkaloids | |
| CN101865924B (en) | Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay | |
| Liu et al. | Development of indirect competitive immunoassay for highly sensitive determination of ractopamine in pork liver samples based on surface plasmon resonance sensor | |
| Vinogradova et al. | Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements | |
| Park et al. | Development of a chemiluminescent immunosensor for chloramphenicol | |
| Zhou et al. | Gold nanoparticle probe-based immunoassay as a new tool for tetrodotoxin detection in puffer fish tissues | |
| CN103439514B (en) | Pesticide and veterinary drug multi-residue detection method based on microarray detection chip | |
| EA011656B1 (en) | In vitro method for simultaneously detecting and identifying antibiotics of different classes and corresponding diagnostic kit | |
| CN101603961A (en) | A kind of light-induced chemiluminescent immunoassay kit of chloromycetin and detection method thereof | |
| CN110567952B (en) | Aptamer-modified nanogold-based cyromazine detection method | |
| Wang et al. | Simultaneous determination of 10 kinds of biogenic amines in rat plasma using high‐performance liquid chromatography coupled with fluorescence detection | |
| Lippolis et al. | Fluorescence polarisation immunoassays for rapid, accurate and sensitive determination of mycotoxins | |
| Longobardi et al. | Determination of ochratoxin A in wine by means of immunoaffinity and aminopropyl solid-phase column cleanup and fluorometric detection | |
| Zhai et al. | Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence | |
| CA2570519A1 (en) | Analyte detection system | |
| Wittmann et al. | Quantitative analysis of 2, 4-dichlorophenoxyacetic acid in water samples by two immunosensing methods | |
| Zhang et al. | Fluorescence polarisation immunoassay based on a monoclonal antibody for the detection of sulphamethazine in chicken muscle | |
| CN101398432B (en) | Light induced chemiluminescent immune assay determination kit for zearalenone and detection method thereof | |
| CN101398425A (en) | Kit for detecting ocean incretion harass chemical substance, preparation method and application | |
| CN113340863A (en) | Enzyme-free circulating amplification aptamer sensor and preparation method and application thereof | |
| Huet et al. | Screening methods and recent developments in the detection of anticoccidials |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200512 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |