CN112578118B - Time-resolved fluorescence immunochromatography kit for rapidly detecting antibiotics - Google Patents
Time-resolved fluorescence immunochromatography kit for rapidly detecting antibiotics Download PDFInfo
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- CN112578118B CN112578118B CN202011310728.6A CN202011310728A CN112578118B CN 112578118 B CN112578118 B CN 112578118B CN 202011310728 A CN202011310728 A CN 202011310728A CN 112578118 B CN112578118 B CN 112578118B
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- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 50
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 49
- 238000003317 immunochromatography Methods 0.000 title abstract description 4
- 238000001514 detection method Methods 0.000 claims abstract description 67
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 63
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 60
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 60
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 53
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 52
- 239000012528 membrane Substances 0.000 claims abstract description 52
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 51
- 239000004005 microsphere Substances 0.000 claims abstract description 33
- 230000027455 binding Effects 0.000 claims abstract description 32
- 239000000706 filtrate Substances 0.000 claims abstract description 29
- 238000010521 absorption reaction Methods 0.000 claims abstract description 28
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 25
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 25
- 239000003306 quinoline derived antiinfective agent Substances 0.000 claims abstract description 23
- 229920003023 plastic Polymers 0.000 claims abstract description 20
- 238000003908 quality control method Methods 0.000 claims abstract description 20
- 239000000427 antigen Substances 0.000 claims abstract description 16
- 108091007433 antigens Proteins 0.000 claims abstract description 16
- 102000036639 antigens Human genes 0.000 claims abstract description 16
- 241000283707 Capra Species 0.000 claims abstract description 15
- 239000013592 cell lysate Substances 0.000 claims abstract description 14
- 229940124036 Hydrolase inhibitor Drugs 0.000 claims abstract description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
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- 102000053602 DNA Human genes 0.000 claims description 39
- 108020004414 DNA Proteins 0.000 claims description 39
- 229940124307 fluoroquinolone Drugs 0.000 claims description 38
- 230000009870 specific binding Effects 0.000 claims description 32
- 230000003115 biocidal effect Effects 0.000 claims description 31
- 239000007788 liquid Substances 0.000 claims description 28
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 claims description 25
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- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
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- 108050001175 Connexin Proteins 0.000 claims description 6
- 230000005847 immunogenicity Effects 0.000 claims description 6
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- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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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
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention discloses a time-resolved fluorescence immunochromatography kit for rapidly detecting antibiotics, which comprises the following components: the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic clamping shell, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane; wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line; the extraction solution comprises a cell lysate, a nucleic acid hydrolysate, a filter column and a filtrate, wherein the filtrate comprises a nucleic acid hydrolase inhibitor. The invention has the characteristics of high detection sensitivity, strong specificity, quick detection and the like.
Description
Technical Field
The invention belongs to the field of antibiotic detection. More particularly, the invention relates to a time-resolved fluorescence immunochromatography kit for rapidly detecting antibiotics.
Background
Fluoroquinolone antibiotics are artificially synthesized broad-spectrum antibacterial agents, and have good sterilization effects, such as escherichia coli, salmonella, klebsiella pneumoniae, brucella, pasteurella multocida, actinobacillus pleuropneumoniae, erysipelas bacillus, proteus, corynebacterium suppurative, bordetella septicum and the like. Thus, fluoroquinolone antibiotics have gained good popularity in animal husbandry. The action mechanism of fluoroquinolone antibiotics is to inhibit bacterial ssDNA gyrase, block ssDNA replication and exert rapid sterilization.
With the expansion of the application range of fluoroquinolone antibiotics, the problem of residues in animal foods is gradually attracting attention. The harm of fluoroquinolone veterinary drug residues in animal foods is mainly represented by: on the one hand, when people use animal foods with a certain residual amount of fluoroquinolones, the residual dose can enter the digestive system of the human body and cause damage to related organs; on the other hand, after people ingest animal food containing fluoroquinolones for a long time, the residual medicinal components in the fluoroquinolones can induce various pathogenic bacteria of human bodies to generate drug resistance, so that the fluoroquinolones have a blocking effect on the application of the fluoroquinolones in human antibacterial treatment. Therefore, the detection of fluoroquinolone veterinary drug residues in animal foods is of great significance.
Disclosure of Invention
It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.
It is still another object of the present invention to provide a time-resolved fluoroimmunochromatographic kit for rapidly detecting antibiotics, which can rapidly and accurately detect fluoroquinolone antibiotics remaining in animal foods.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a time-resolved fluoroimmunochromatographic kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
According to the invention, the ssDNA sequence with specific binding fluoroquinolone antibiotics is connected to the protein, and the Eu fluorescent microspheres are marked on the protein, so that under the action of excitation light, the specific fluoroquinolone antibiotics binding nucleic acid protein compound marked by the Eu fluorescent microspheres emits stable fluorescence, and the detection of residual fluoroquinolone antibiotics in animal foods is realized.
Preferably, the olfactorin is chemically modified or non-covalently linked with TYLCV-C1 protein to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain the protein nucleic acid complex. The invention takes TYLCV-C1 protein as a connecting junction of the olfactory protein and the antibiotic specific binding ssDNA, so as to realize the connection of the antibiotic specific binding ssDNA to the olfactory protein; the olfactory protein has the function of enhancing Eu fluorescence, and Eu fluorescent microspheres are marked on the olfactory protein, so that the detection lower limit of the invention is further enhanced through the fluorescence enhancement function of the olfactory protein.
Preferably, the sequence of the antibiotic that specifically binds ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3'79. The ssDNA sequence has good stability, high sensitivity in combination with fluoroquinolone antibiotics, and short ssDNA sequence, can be quickly synthesized artificially, and has low preparation cost.
Preferably, the fluoroquinolone antibiotics and bovine serum albumin are used for preparing the whole antigen with immunogenicity and reactivity, the coupling ratio is 1:100, and the fluoroquinolone antibiotics and bovine serum albumin compound is obtained after coupling and dialysis purification.
Preferably, the fluoroquinolone antibiotics bovine serum albumin complex is diluted to 1mg/mL by PBS-sucrose buffer solution to prepare detection T line liquid, and a point film machine is used for spraying the detection T line liquid on a nitrocellulose film to prepare a detection T line; diluting goat anti-rabbit secondary antibody with PBS-sucrose buffer solution to 1mg/mL to obtain detection C line liquid, spraying point detection C line liquid on a nitrocellulose membrane by a point film machine, and preparing detection C line; and drying the nitrocellulose membrane after spraying at a low temperature.
Preferably, the cell lysate comprises the following components in percentage by mass: triton X-100%, SDS 1%, naCl 5%; the nucleic acid hydrolysis liquid is 200U/L of nucleic acid hydrolase.
Preferably, the method of using the kit comprises the steps of:
1) Weighing 0.5-1g of tissue to be measured, placing the tissue into a homogenizing pipe, adding 1mL of extracting solution into the homogenizing pipe, and homogenizing to obtain homogenate;
2) Placing the homogenate at the upper end of the filtering column, centrifuging to separate filter residues from filtrate, adding 1mL of filtrate into the upper end of the filtering column again, centrifuging, collecting filtrate, and uniformly mixing to obtain liquid to be detected;
3) Dropping the liquid to be detected into a sample pad, reading the fluorescent intensity of a T line and the fluorescent intensity of a C line by using a POTC analyzer after 5min, calculating a T/C value, and calculating the concentration of fluoroquinolone antibiotics in the sample according to the prepared standard curve.
Preferably, the fluoroquinolone antibiotic is ofloxacin.
The invention at least comprises the following beneficial effects:
1. according to the invention, the ssDNA sequence with specific binding fluoroquinolone antibiotics is connected to the protein, and the Eu fluorescent microspheres are marked on the protein, so that under the action of excitation light, the specific fluoroquinolone antibiotics binding nucleic acid protein compound marked by the Eu fluorescent microspheres emits stable fluorescence, and the detection of residual fluoroquinolone antibiotics in animal foods is realized.
2. The invention takes TYLCV-C1 protein as a connecting junction of the olfactory protein and the antibiotic specific binding ssDNA, so as to realize the connection of the antibiotic specific binding ssDNA to the olfactory protein; the olfactory protein has the function of enhancing Eu fluorescence, and Eu fluorescent microspheres are marked on the olfactory protein, so that the detection lower limit of the invention is further enhanced through the fluorescence enhancement function of the olfactory protein.
3. The ssDNA sequence has good stability and high sensitivity of combining with fluoroquinolone antibiotics, is shorter, can be quickly synthesized artificially, and has low preparation cost.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic representation of fluorescence of different substances according to the invention;
FIG. 2 illustrates fluorescence graphs of specific fluoroquinolone antibiotic-binding nucleic acid protein complexes labeled with Eu fluorescent microspheres at different concentrations.
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention by reference to the specification.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The TYLCV-C1 protein sequence used in the invention is the same as the sequence in application number 201910730507.5;
the olfactory proteins used in the present invention are purchased from sigma company;
the Eu fluorescent microspheres used in the present invention are purchased from Basil biological medicine Co.
Example 1
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
According to the invention, the ssDNA sequence with specific binding fluoroquinolone antibiotics is connected to the protein, and the Eu fluorescent microspheres are marked on the protein, so that under the action of excitation light, the specific fluoroquinolone antibiotics binding nucleic acid protein compound marked by the Eu fluorescent microspheres emits stable fluorescence, and the detection of residual fluoroquinolone antibiotics in animal foods is realized.
Example 2
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein and TYLCV-C1 protein are connected through chemical modification to obtain connexin, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connexin is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain the protein nucleic acid complex.
The invention takes TYLCV-C1 protein as a connecting junction of the olfactory protein and the antibiotic specific binding ssDNA, so as to realize the connection of the antibiotic specific binding ssDNA to the olfactory protein; the olfactory protein has the function of enhancing Eu fluorescence, and Eu fluorescent microspheres are marked on the olfactory protein, so that the detection lower limit of the invention is further enhanced through the fluorescence enhancement function of the olfactory protein.
Example 3
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein is connected with TYLCV-C1 protein through chemical modification to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain a protein nucleic acid compound;
the sequences of antibiotic specific binding ssDNA are:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
the ssDNA sequence has good stability, high sensitivity in combination with fluoroquinolone antibiotics, and short ssDNA sequence, can be quickly synthesized artificially, and has low preparation cost.
Example 4
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line; preparing complete antigen with immunogenicity and reactivity by using fluoroquinolone antibiotics and bovine serum albumin, wherein the coupling ratio is 1:100, and dialyzing and purifying after coupling to obtain fluoroquinolone antibiotics bovine serum albumin compound;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein is connected with TYLCV-C1 protein through chemical modification to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain a protein nucleic acid compound;
the sequences of antibiotic specific binding ssDNA are:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 5
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line; preparing complete antigen with immunogenicity and reactivity by using fluoroquinolone antibiotics and bovine serum albumin, wherein the coupling ratio is 1:100, and dialyzing and purifying after coupling to obtain fluoroquinolone antibiotics bovine serum albumin compound; diluting the fluoroquinolone antibiotic bovine serum albumin complex to 1mg/mL by using PBS-sucrose buffer solution to prepare detection T line liquid, and spraying the detection T line liquid on a nitrocellulose membrane by using a point film machine to prepare a detection T line; diluting goat anti-rabbit secondary antibody with PBS-sucrose buffer solution to 1mg/mL to obtain detection C line liquid, spraying point detection C line liquid on a nitrocellulose membrane by a point film machine, and preparing detection C line; drying the nitrocellulose membrane after spraying at a low temperature;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate,
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein is connected with TYLCV-C1 protein through chemical modification to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain a protein nucleic acid compound;
the sequences of antibiotic specific binding ssDNA are:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 6
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line; preparing complete antigen with immunogenicity and reactivity by using fluoroquinolone antibiotics and bovine serum albumin, wherein the coupling ratio is 1:100, and dialyzing and purifying after coupling to obtain fluoroquinolone antibiotics bovine serum albumin compound; diluting the fluoroquinolone antibiotic bovine serum albumin complex to 1mg/mL by using PBS-sucrose buffer solution to prepare detection T line liquid, and spraying the detection T line liquid on a nitrocellulose membrane by using a point film machine to prepare a detection T line; diluting goat anti-rabbit secondary antibody with PBS-sucrose buffer solution to 1mg/mL to obtain detection C line liquid, spraying point detection C line liquid on a nitrocellulose membrane by a point film machine, and preparing detection C line; drying the nitrocellulose membrane after spraying at a low temperature;
the extracting solution comprises a cell lysate and a nucleic acid hydrolysate, wherein the cell lysate comprises the following components in percentage by mass: triton X-100%, SDS 1%, naCl 5%; the nucleic acid hydrolysis liquid is 200U/L of nucleic acid hydrolase;
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein is connected with TYLCV-C1 protein through chemical modification to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain a protein nucleic acid compound;
the sequences of antibiotic specific binding ssDNA are:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 7
A time-resolved fluoroimmunoassay kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the detection method comprises the steps of adsorbing a specific ofloxacin binding nucleic acid protein complex and a rabbit IgG antibody marked by Eu fluorescent microspheres on a binding pad, detecting a bovine serum albumin complex coated with ofloxacin antigen on a T line, and coating a goat anti-rabbit secondary antibody on a quality control C line; preparing complete antigen with immunogenicity and reactivity by ofloxacin and bovine serum albumin, wherein the coupling ratio is 1:100, and dialyzing and purifying after coupling to obtain ofloxacin Sha Xingniu serum albumin compound; diluting the oxyfluoride Sha Xingniu serum protein complex with PBS-sucrose buffer solution to 1mg/mL to obtain a detection T line solution, and spraying the detection T line solution on a nitrocellulose membrane by a spot film machine to prepare a detection T line; diluting goat anti-rabbit secondary antibody with PBS-sucrose buffer solution to 1mg/mL to obtain detection C line liquid, spraying point detection C line liquid on a nitrocellulose membrane by a point film machine, and preparing detection C line; drying the nitrocellulose membrane after spraying at a low temperature;
the extracting solution comprises a cell lysate and a nucleic acid hydrolysate, wherein the cell lysate comprises the following components in percentage by mass: triton X-100%, SDS 1%, naCl 5%; the nucleic acid hydrolysis liquid is 200U/L of nucleic acid hydrolase;
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
the olfactory protein is connected with TYLCV-C1 protein through chemical modification to obtain a connecting protein, the 5' end of the antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connecting protein is connected with the antibiotic specific binding ssDNA, manganese ion with the final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain a protein nucleic acid compound;
the sequences of antibiotic specific binding ssDNA are:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 8
The application method of the kit prepared by the invention comprises the following steps:
1) Weighing 0.5g of tissue to be measured, placing the tissue into a homogenizing pipe, adding 1mL of extracting solution into the homogenizing pipe, and homogenizing to obtain homogenate;
2) Placing the homogenate at the upper end of the filtering column, centrifuging to separate filter residues from filtrate, adding 1mL of filtrate into the upper end of the filtering column again, centrifuging, and collecting filtrate to obtain liquid to be detected;
3) Dropping the liquid to be detected into a sample pad, reading the fluorescent intensity of a T line and the fluorescent intensity of a C line by using a POTC analyzer after 5min, calculating a T/C value, and calculating the concentration of ofloxacin in the sample according to a prepared standard curve.
1. Specific binding assay
Dissolving 1uL of Eu fluorescent microsphere marked specific ofloxacin combined nucleic acid protein complex with the concentration of 50umol/L in 198uL of PBS buffer solution, adding 1uL of 10umol/L of ofloxacin, uniformly mixing, adding a protein precipitating agent to precipitate the Eu fluorescent microsphere marked specific ofloxacin combined nucleic acid protein complex, centrifuging, collecting supernatant, measuring the ofloxacin content in the supernatant by using conventional liquid chromatography, and repeating the test for 3 times, wherein the result shows that the ofloxacin content in the supernatant is not detected. The Eu fluorescent microsphere marked specific ofloxacin binding nucleic acid protein compound prepared by the invention can be specifically bound with ofloxacin.
Dissolving 100uL,50umol/L of Eu fluorescent microsphere marked specific ofloxacin binding nucleic acid protein complex in 19800uL of PBS buffer solution, adding 100uL,10umol/L of ofloxacin, uniformly mixing, adding a protein precipitating agent to precipitate Eu fluorescent microsphere marked specific ofloxacin binding nucleic acid protein complex, centrifuging, collecting supernatant, concentrating the supernatant to 20 times of original, measuring the ofloxacin content in the concentrated solution by using conventional liquid chromatography, and repeating the test for 3 times, wherein the result shows that the ofloxacin content in the concentrated solution is 0.13ug/L.
2. Fluorescence enhancement test
1uL 8mg/L Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein complex is dissolved in 999uL PBS buffer, and the emission spectrum is shown in figure 1 under the action of the excitation wavelength of 270 nm.
1uL of 8mg/L Eu fluorescent microspheres are dissolved in 999uL of PBS buffer solution, and the emission spectrum is shown in figure 1 under the excitation wavelength of 270 nm.
1uL 8mg/L of the connexin was dissolved in 999uL of PBS buffer and the emission spectrum was as shown in FIG. 1 under the excitation wavelength of 270 nm.
In FIG. 1, 1 is Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein complex; 2 is Eu fluorescent microsphere; 3 is a connexin.
The result shows that the fluorescence intensity of the specific ofloxacin binding nucleic acid protein complex marked by the Eu fluorescent microsphere is obviously enhanced.
3. Determination of quantitative Standard Curve
The Eu fluorescent microsphere-labeled specific ofloxacin binding nucleic acid protein complex standard is diluted into 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.4ng/mL, 0.8ng/mL, 1.0ng/mL and 2.0ng/mL respectively by using PBS buffer, each concentration is detected 10 times according to a standard operation method, the fluorescence intensity is taken as an ordinate, the Eu fluorescent microsphere-labeled specific ofloxacin binding nucleic acid protein complex is taken as an abscissa, and a standard curve is obtained, and the result is shown in FIG. 2.
4. Accuracy verification test
The concentration of the ofloxacin solution with the concentration of 0.20ug/L is detected by the kit (example 7) and the liquid chromatography respectively, the test is repeated three times, the average concentration of the detection of the kit is 0.199ug/L, and the concentration of the detection of the liquid chromatography is 0.21ug/L, which shows that the detection accuracy of the kit is higher.
Although embodiments of the invention have been disclosed above, they are not limited to the use listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. Therefore, the invention is not to be limited to the specific details disclosed herein without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (6)
1. A time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic clamping shell, wherein a PVC plate, a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad are arranged in the plastic clamping shell in an interlayer manner, the sample pad, the bonding pad, the nitrocellulose membrane and the absorption pad are sequentially and lapped and assembled on the PVC plate, the absorption pad and the bonding pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the bonding pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
wherein, the specific fluoroquinolone antibiotic binding nucleic acid protein complex and rabbit IgG antibody marked by Eu fluorescent microspheres are adsorbed on the binding pad, the fluoroquinolone antibiotic antigen bovine serum albumin complex coated on the T line is detected, and the goat anti-rabbit secondary antibody is coated on the quality control C line;
the olfactorin and TYLCV-C1 protein are connected through chemical modification or non-covalent bond to obtain connexin, 5' end of antibiotic specific binding ssDNA is connected with cgtataatatta sequence, the connexin is connected with antibiotic specific binding ssDNA, manganese ion with final concentration of 6mM is added, and the reaction is carried out for 10min at 25 ℃ to obtain protein nucleic acid complex;
the sequences of antibiotic specific binding ssDNA are:
5’ -TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’ 79;
an extracting solution, wherein the extracting solution comprises a cell lysate and a nucleic acid hydrolysate;
a filtration column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
2. The kit for rapid antibiotic detection according to claim 1, wherein the fluoroquinolone antibiotics and bovine serum albumin are prepared into total antigens with immunogenicity and reactivity, the coupling ratio is 1:100, and the fluoroquinolone antibiotics and bovine serum albumin complexes are obtained by dialysis and purification after coupling.
3. The time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics according to claim 1, wherein fluoroquinolone antibiotics bovine serum albumin complex is diluted to 1mg/mL with PBS-sucrose buffer solution to prepare a detection T line liquid, and the detection T line liquid is sprayed on a nitrocellulose membrane by a point film machine to prepare a detection T line; diluting goat anti-rabbit secondary antibody with PBS-sucrose buffer solution to 1mg/mL to obtain detection C line liquid, spraying point detection C line liquid on a nitrocellulose membrane by a point film machine, and preparing detection C line; and drying the nitrocellulose membrane after spraying at a low temperature.
4. The time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics according to claim 1, in which the cell lysate comprises the following components by mass fraction: triton X-100%, SDS 1%, naCl 5%; the nucleic acid hydrolysis liquid is 200U/L of nucleic acid hydrolase.
5. The time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics according to any one of claims 1 to 4, in which the method of use thereof comprises the steps of:
1) Weighing 0.5-1g of tissue to be measured, placing the tissue into a homogenizing pipe, adding 1mL of extracting solution into the homogenizing pipe, and homogenizing to obtain homogenate;
2) Placing the homogenate at the upper end of a filtering column, centrifuging to separate filter residues from filtrate, adding 1mL of filtrate into the upper end of the filtering column again, centrifuging, collecting filtrate, and uniformly mixing to obtain liquid to be detected;
3) Dropping the liquid to be detected into a sample pad, reading the fluorescent intensity of a T line and the fluorescent intensity of a C line by using a POTC analyzer after 5min, calculating a T/C value, and calculating the concentration of fluoroquinolone antibiotics in the sample according to the prepared standard curve.
6. The time-resolved fluoroimmunochromatographic kit for rapid detection of antibiotics according to claim 1, in which the fluoroquinolone antibiotic is ofloxacin.
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