CN110823846A - Application of novel fluorescent dye AccuBlue in detection of aptamer - Google Patents
Application of novel fluorescent dye AccuBlue in detection of aptamer Download PDFInfo
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- 102000053602 DNA Human genes 0.000 claims abstract description 32
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 4
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- 231100000765 toxin Toxicity 0.000 claims abstract description 3
- 239000000975 dye Substances 0.000 claims description 10
- 239000013076 target substance Substances 0.000 claims description 7
- 230000000295 complement effect Effects 0.000 claims description 6
- 108020004635 Complementary DNA Proteins 0.000 claims description 4
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- 238000010804 cDNA synthesis Methods 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 4
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 229960005305 adenosine Drugs 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 229940088710 antibiotic agent Drugs 0.000 claims description 2
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention relates to application of a novel fluorescent dye AccuBlue in a nucleic acid aptamer detection method. The characteristic that the fluorescent dye AccuBlue only emits weak fluorescence when in a free state or coexists with single-stranded DNA (deoxyribonucleic acid), and can be combined with double-stranded DNA when coexisting with the double-stranded DNA so that a fluorescence signal is obviously enhanced is combined with the characteristics of high affinity and high specificity of the aptamer, and the nucleic acid aptamer detection method based on the AccuBlue is established. Meanwhile, because the AccuBlue does not have membrane penetrability, can not be identified with DNA in cell nuclei, and can not generate interference reaction, the method can be applied to multiple fields of biological detection, food safety, pollutant analysis, pathogenic bacteria detection, medicine research and the like, and can be used for efficiently, quickly and strongly detecting inorganic organic molecules, proteins, pathogenic bacteria, biotoxins and the like. The method overcomes the defects of high manufacturing cost, large interference and limited application range in the prior art, and has the advantages of simplicity, convenience, wide application range, high sensitivity, low manufacturing cost, economy and applicability.
Description
Technical Field
The invention relates to application of a novel fluorescent dye AccuBlue in a nucleic acid aptamer detection method, and belongs to the field of analysis and detection.
Background
The detection method of the aptamer is a hot spot and a focus of research in the detection method of foods and medicines in recent years. Aptamers (aptamers) are single-stranded nucleotides (DNA or RNA) that are capable of binding to a target substance, such as a drug, an inorganic or organic molecule, a protein, or the like, with high affinity and specificity. Usually consists of 10 to 100 bases. Due to the diversity of the spatial structure of the single-stranded nucleotide, the single-stranded nucleotide forms a G-quadruplex, a bulge loop and other complex tertiary structures by complementary pairing of bases in a strand and hydrogen bond action. The aptamer can be combined with a target in a high specificity and high affinity manner, the aptamer can be labeled by various markers, and after the aptamer is connected with a biosensor, the aptamer detection method can be applied to basic research, analysis and detection, and research and development of medicines and clinical diagnosis. In the detection method of the aptamer, the aptamer is modified by nano substances, fluorescent substances, related enzyme materials, biotin and proteins with specific purposes, and then the detection is carried out by enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence method and the like.
In recent years, many fluorescence sensors based on aptamers have been reported, but few studies have been made on fluorescent dyes having wide applicability. The dyes such as PicoGreen, SYBR Green and the like which are commonly used are found to have the characteristic that only weak fluorescence is emitted under the condition of free or coexisting with single-stranded DNA (deoxyribonucleic acid), and the fluorescence intensity is obviously enhanced when the dyes coexist with double-stranded DNA. However, both of the above dyes have membrane permeability, and when the target is bacteria or cells, they bind to DNA in nuclei, thus causing great interference with the detection result, and thus are not suitable for detection of bacteria or cells.
Therefore, the invention provides a novel fluorescent dye AccuBlue, which not only has the characteristic that the fluorescent dye only emits weak fluorescence when coexisting with single-stranded DNA and can be combined with double-stranded DNA to obviously enhance a fluorescent signal when coexisting with the double-stranded DNA, but also has no membrane penetrability so as not to be identified with the DNA in a cell nucleus, and therefore, the fluorescent dye can be widely applied to the fluorescence detection of nucleic acid aptamers for constructing different substances. The establishment of the method can provide a fast, convenient, economic and efficient feasible detection mode for the fields of biological detection, food safety, pollutant analysis, pathogenic bacteria detection, medicine research and the like, and open a new door in the detection field.
In order to more clearly introduce the detection method related to the patent, the invention takes the detection of enrofloxacin in animal-derived food as an example for experiment, and the experimental result shows that the linear range of the detection of enrofloxacin residue in the animal-derived food (milk powder, beef and shrimp meat) is 20-1000 mug/L, and the detection limit is 9.96 mug/L. The detection method can be used for detecting enrofloxacin. The method has the advantages of economy, rapidness, sensitivity, accuracy and the like due to the utilization of the novel fluorescent dye AccuBlue, can be widely used for detecting various food hazards, and provides a new idea and method for guaranteeing food safety.
Disclosure of Invention
The invention aims to provide a novel fluorescent dye which can be applied to a nucleic acid aptamer detection method. The dye can be combined with a nucleic acid aptamer with high affinity and high specificity, and is used for detecting various substances.
In order to achieve the above object, the present invention is achieved by the following means. The method is characterized in that:
after incubating the aptamer and the target substance for a period of time at room temperature, an aptamer-target substance complex is formed, and then DNA complementary to the aptamer and an AccuBlue dye are added to the complex, so that free aptamer which cannot be bound with the target substance can be hybridized with the complementary DNA to form double-stranded DNA (dsDNA), and the AccuBlue dye can be inserted into the dsDNA to cause a remarkable increase of a fluorescence signal. Theoretically, the higher the concentration of the target, the less free aptamers in the system that are not bound to the target, and thus the less dsDNA that can be hybridized with complementary DNA, the less AccuBlue dye that is inserted into the dsDNA, and thus the smaller the fluorescent signal, thereby allowing quantitative analysis.
Wherein the aptamer includes, but is not limited to, artificial synthesis or single stranded after PCR amplification.
Such targets include, but are not limited to, proteins, organics, biological contaminants, antibiotics, adenosine, toxins, pathogenic bacteria, and the like.
The invention has the advantages that:
the aptamer in the detection method can be changed due to the change of the target object, the source of the aptamer can be customized or screened according to the requirements of users, and the detection method has flexible and variable range.
AccuBlue as a membrane-free penetrating fluorescent dye can be combined with a nucleic acid aptamer with high affinity, is not only suitable for detecting proteins, organic molecules, inorganic molecules and the like, but also can be applied to detecting cells or pathogenic bacteria and the like, and the detection method has wider applicability.
The detection method of the nucleic acid aptamer based on AccuBlue is simple, convenient, wide in application range, high in sensitivity, low in manufacturing cost, economical and applicable.
Drawings
The invention will be described in more detail with reference to the accompanying drawings
FIG. 1 is a schematic diagram for detecting enrofloxacin based on aptamer and fluorescent dye AccuBlue
FIG. 2 is a linear curve chart of the method for detecting enrofloxacin with different concentrations and fluorescence change
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
The following examples embody the methods of operation of the present invention, but are not intended to limit the invention thereto.
Example 1: application of detection method in detecting enrofloxacin
Preparing enrofloxacin into 1g/L ENR mother liquor for later use. Then diluted into solutions with mass concentrations of 0, 20, 50, 100, 500, 1000 and 2000. mu.g/L, respectively. And (3) uniformly mixing 10 mu L of ENR solution with different concentrations and 10 mu L of 10nmol/L ENR aptamer-2 solution in a hole, incubating for 10min at 25 ℃, adding 10 mu L of 10nmol/L complementary strand DNA-2 solution, continuing to react for 10min at 25 ℃, adding 200 mu L of AccuBlue solution, and detecting the fluorescence intensity. When the amount of ENR aptamer is constant, the higher the ENR concentration is, the fewer aptamers capable of binding to complementary strands in the system are, the less dsDNA is formed, and the fluorescence intensity F generated by recognition of dsDNA by AccuBlueThe smaller the value. In the absence of ENR drug in the solution, the aptamer was fully bound to its corresponding complementary strand, and fluorescence intensity F0Maximum, F0Is constant, F/F0Is proportional to the magnitude of the F value, the larger the F, the F/F0The larger. Therefore, under the optimal experimental conditions, the standard curve of the method is Y = -0.1868x +1.186 (as in FIG. 2), R2= 0.9457. The detection limit is 9.96 mu g/L, and the linear range is 20-1000 mu g/L.
In the method for detecting enrofloxacin in animal-derived food by using the aptamer based on the novel fluorescent dye AccuBlue, the sequence of the enrofloxacin aptamer is as follows:
5’- CCCATCAGGGGGCTAGGCTAACACGGTTCGGCTCTCTGAGCCCGGGTTATT
TCAGGGGGA-3'; synthesized by Biotechnology engineering (Shanghai) Inc.
Example 2: application of detection method in detecting enrofloxacin in milk powder, shrimp meat and beef
Milk powder: 5.0g of skim milk powder was weighed, 10mL of 1 XPBS was added, the mixture was placed in a 25mL centrifuge tube, centrifuged at 14000rpm for 20min, and the supernatant was taken out as a blank sample.
Shrimp and beef: selecting an edible part, chopping and uniformly mixing, accurately weighing 5.0g of meat sample, adding 5.0g of anhydrous sodium sulfate, grinding, uniformly grinding, pouring into a 10mL centrifuge tube, adding an extraction solvent (4 mL of acetonitrile and 0.04 mL of glacial acetic acid), carrying out high-speed vortex for 2min, centrifuging at 3000 rpm for 12min, and taking out a supernatant; repeating the above operation on the residue for another 1 time, combining the supernatants, drying with nitrogen, and dissolving the residue with Tris buffer solution for later use.
Adding enrofloxacin with final concentration of 200 mug/kg, 500 mug/kg, 1000 mug/kg and each concentration into blank samples to perform 3 parallels, and measuring fluorescence intensity value after processing by an optimized method, wherein each measurement is repeated for 3 times. And calculating the addition recovery rate according to a formula. The results are shown in Table 1.
TABLE 1 results of recovery of samples by fluorimetry with a standard (n = 3; μ g/kg)
Claims (5)
1. The application of the novel fluorescent dye AccuBlue in detection of the aptamer is characterized by comprising the following contents: after incubating the aptamer and the target substance for a period of time at room temperature, an aptamer-target substance complex is formed, and then DNA complementary to the aptamer and an AccuBlue dye are added to the complex, so that free aptamer which cannot be bound with the target substance can be hybridized with the complementary DNA to form double-stranded DNA (dsDNA), and the AccuBlue dye can be inserted into the dsDNA to cause a remarkable increase of a fluorescence signal.
2. Theoretically, the higher the concentration of the target, the less free aptamers in the system that are not bound to the target, and thus the less dsDNA that can be hybridized with complementary DNA, the less AccuBlue dye that is inserted into the dsDNA, and thus the smaller the fluorescent signal, thereby allowing quantitative analysis.
3. The use of the novel fluorescent dye AccuBlue according to claim 1 in the detection of aptamers, wherein:
(1) the novel fluorescent dye AccuBlue only emits weak fluorescence under the condition of free or coexisting with single-stranded DNA (deoxyribonucleic acid), and has the characteristic of obviously enhancing the fluorescence intensity when coexisting with double-stranded DNA (deoxyribonucleic acid), and does not have membrane penetrability.
(2) wherein the aptamer includes, but is not limited to, synthetic or single stranded after PCR amplification.
(3) the target includes, but is not limited to, proteins, organisms, biological contaminants, antibiotics, adenosine, toxins, pathogens, and the like.
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| CN113481206A (en) * | 2021-09-03 | 2021-10-08 | 中国农业大学 | Rapid detection method of enrofloxacin |
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| CN103808701A (en) * | 2013-09-18 | 2014-05-21 | 河南省农业科学院 | Homogeneous-phase rapid detection method of ochratoxin A based on nucleic acid chimeric dye fluorescence quenching |
| 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 |
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