CN112578118A - 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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- CN112578118A CN112578118A CN202011310728.6A CN202011310728A CN112578118A CN 112578118 A CN112578118 A CN 112578118A CN 202011310728 A CN202011310728 A CN 202011310728A CN 112578118 A CN112578118 A CN 112578118A
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- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 48
- 229940088710 antibiotic agent Drugs 0.000 title claims abstract description 47
- 238000003317 immunochromatography Methods 0.000 title claims description 3
- 238000001514 detection method Methods 0.000 claims abstract description 72
- 239000012528 membrane Substances 0.000 claims abstract description 62
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 54
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- 239000000243 solution Substances 0.000 claims description 47
- 102000053602 DNA Human genes 0.000 claims description 38
- 108020004414 DNA Proteins 0.000 claims description 38
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- 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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Abstract
The invention discloses a time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, which comprises: the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane; the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody; the extracting solution comprises cell lysate, nucleic acid hydrolysate, a filter column and filtrate, wherein the filtrate contains 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 immunochromatographic kit for rapidly detecting antibiotics.
Background
The fluoroquinolone antibiotics are artificial broad-spectrum antibacterial drugs with good sterilization, such as escherichia coli, salmonella, klebsiella pneumoniae, brucella, pasteurella multocida, actinobacillus pleuropneumoniae, erysipelothrix rhusiopathiae, proteus, corynebacterium pyogenes, bordetella septicum and the like. Therefore, fluoroquinolone antibiotics are well popularized 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, people pay more attention to the problem of residue in animal food. The harm of fluoroquinolone veterinary drug residues in animal food is mainly reflected in that: on one hand, when people use animal food with a certain amount of residual fluoroquinolones, the residual dose can enter the digestive system of human bodies and cause damage to related organs; on the other hand, after people take the fluoroquinolone-containing animal food for a long time, the residual medicine components in the fluoroquinolone-containing animal food can induce various pathogenic bacteria of human bodies to generate medicine resistance, and further, the fluoroquinolone-containing animal food has an inhibiting effect on the application of the fluoroquinolone in human antibacterial treatment. Therefore, the detection of fluoroquinolone veterinary drug residues in animal food is of great significance.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
Still another object of the present invention is to provide a time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics, which can rapidly and accurately detect fluoroquinolone antibiotics remaining in animal food.
To achieve these objects and other advantages of the present invention, there is provided a time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
According to the invention, the ssDNA sequence with the specificity-binding fluoroquinolone antibiotics is connected to the protein, and the Eu fluorescent microspheres are marked on the protein, so that the Eu fluorescent microsphere-marked specificity-fluoroquinolone antibiotics-binding nucleic acid protein complex emits stable fluorescence under the action of exciting light, and thus the detection of residual fluoroquinolone antibiotics in animal food is realized.
Preferably, the olfactory protein is chemically modified or non-covalently linked to the TYLCV-C1 protein to obtain a connexin, the 5' end of the antibiotic-specific binding ssDNA is linked to a cgtataatatta sequence, the connexin is linked to the antibiotic-specific binding ssDNA, manganese ions are added to a final concentration of 6mM, and the reaction is carried out at 25 ℃ for 10min to obtain a protein-nucleic acid complex. According to the invention, the TYLCV-C1 protein is used as a junction of olfactory protein and the ssDNA specifically bound by the antibiotic, so that the ssDNA specifically bound by the antibiotic is connected to the olfactory protein; the olfactory protein has the effect of enhancing Eu fluorescence, Eu fluorescent microspheres are marked on the olfactory protein, and the lower limit of detection of the invention is further enhanced through the fluorescence enhancement effect 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 and high sensitivity of combination with fluoroquinolone antibiotics, is short, can be rapidly synthesized manually, and has low preparation cost.
Preferably, the fluoroquinolone antibiotics and bovine serum albumin are used for preparing a complete antigen with immunogenicity and reactivity, the coupling ratio is 1: 100, and the fluoroquinolone antibiotics bovine serum albumin compound is obtained after coupling and dialysis purification.
Preferably, diluting the fluoroquinolone antibiotic bovine serum albumin compound 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 membrane spraying machine to prepare a detection T line; diluting the goat anti-rabbit secondary antibody to 1mg/mL by using PBS-sucrose buffer solution to prepare detection C line solution, and spraying the detection C line solution on a nitrocellulose membrane by using a membrane spraying machine to prepare a detection C line; drying the sprayed nitrocellulose membrane at low temperature.
Preferably, the cell lysate comprises the following components in percentage by mass: triton X-1003%, SDS 1% and NaCl 5%; the nucleic acid hydrolysis liquid is 200U/L nucleic acid hydrolase.
Preferably, the method of using the kit comprises the following steps:
1) weighing 0.5-1g of tissue to be detected, placing the tissue to be detected in a homogenizing tube, adding 1mL of extracting solution into the homogenizing tube, and homogenizing to obtain homogenate;
2) placing the homogenate liquid on the upper end of the filter column, centrifuging to separate filter residue from filtrate, adding 1mL of filtrate to the upper end of the filter column again, centrifuging, collecting filtrate, and mixing uniformly to obtain a liquid to be detected;
3) and (3) dripping the liquid to be detected into a sample pad, reading the fluorescence intensity of the T line and the fluorescence intensity of the C line by using a POTC analyzer after 5min, calculating a T/C value, and calculating the concentration of the 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:
the invention relates to a method for detecting residual fluoroquinolone antibiotics in animal food, which comprises the steps of connecting a ssDNA sequence with specific binding fluoroquinolone antibiotics to protein, and labeling the protein with Eu fluorescent microspheres, so that the specific fluoroquinolone antibiotics labeled by the Eu fluorescent microspheres can emit stable fluorescence in combination with nucleic acid protein under the action of exciting light, thereby realizing the detection of residual fluoroquinolone antibiotics in the animal food.
Secondly, the protein TYLCV-C1 is used as a connecting pivot for the olfactory protein and the ssDNA specifically bound by the antibiotic, so that the ssDNA specifically bound by the antibiotic is connected to the olfactory protein; the olfactory protein has the effect of enhancing Eu fluorescence, Eu fluorescent microspheres are marked on the olfactory protein, and the lower limit of detection of the invention is further enhanced through the fluorescence enhancement effect of the olfactory protein.
The ssDNA sequence of the invention has good stability, high sensitivity of combination with fluoroquinolone antibiotics, shorter ssDNA sequence, rapid artificial synthesis and 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 the fluorescence of various substances of the present invention;
FIG. 2 illustrates the fluorescence curves 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 with reference to the description.
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 sequence of the TYLCV-C1 protein used in the invention is the same as the sequence in application No. 201910730507.5;
the sniff protein used in the present invention was purchased from sigma;
the Eu fluorescent microsphere used in the invention is purchased from egg-based biological medicine, Inc.
Example 1
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
According to the invention, the ssDNA sequence with the specificity-binding fluoroquinolone antibiotics is connected to the protein, and the Eu fluorescent microspheres are marked on the protein, so that the Eu fluorescent microsphere-marked specificity-fluoroquinolone antibiotics-binding nucleic acid protein complex emits stable fluorescence under the action of exciting light, and thus the detection of residual fluoroquinolone antibiotics in animal food is realized.
Example 2
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin and the antibiotic specific binding ssDNA, adding manganese ions with the final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex.
According to the invention, the TYLCV-C1 protein is used as a junction of olfactory protein and the ssDNA specifically bound by the antibiotic, so that the ssDNA specifically bound by the antibiotic is connected to the olfactory protein; the olfactory protein has the effect of enhancing Eu fluorescence, Eu fluorescent microspheres are marked on the olfactory protein, and the lower limit of detection of the invention is further enhanced through the fluorescence enhancement effect of the olfactory protein.
Example 3
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin with the antibiotic specific binding ssDNA, adding manganese ions with final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex;
the sequence of antibiotic specific binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
the ssDNA sequence has good stability and high sensitivity of combination with fluoroquinolone antibiotics, is short, can be rapidly synthesized manually, and has low preparation cost.
Example 4
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody; preparing a full 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 a fluoroquinolone antibiotic bovine serum albumin compound;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin with the antibiotic specific binding ssDNA, adding manganese ions with final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex;
the sequence of antibiotic specific binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 5
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody; preparing a full 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 a fluoroquinolone antibiotic bovine serum albumin compound; diluting the fluoroquinolone antibiotic bovine serum albumin compound to 1mg/mL by using PBS-sucrose buffer solution to prepare detection T line solution, and spraying the detection T line solution on a nitrocellulose membrane by using a membrane dispenser to prepare a detection T line; diluting the goat anti-rabbit secondary antibody to 1mg/mL by using PBS-sucrose buffer solution to prepare detection C line solution, and spraying the detection C line solution on a nitrocellulose membrane by using a membrane spraying machine to prepare a detection C line; drying the sprayed nitrocellulose membrane at a low temperature;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin with the antibiotic specific binding ssDNA, adding manganese ions with final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex;
the sequence of antibiotic specific binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 6
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody; preparing a full 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 a fluoroquinolone antibiotic bovine serum albumin compound; diluting the fluoroquinolone antibiotic bovine serum albumin compound to 1mg/mL by using PBS-sucrose buffer solution to prepare detection T line solution, and spraying the detection T line solution on a nitrocellulose membrane by using a membrane dispenser to prepare a detection T line; diluting the goat anti-rabbit secondary antibody to 1mg/mL by using PBS-sucrose buffer solution to prepare detection C line solution, and spraying the detection C line solution on a nitrocellulose membrane by using a membrane spraying machine to prepare a detection C line; drying the sprayed nitrocellulose membrane at a low temperature;
the cell lysis solution comprises the following components in percentage by mass: triton X-1003%, SDS 1% and NaCl 5%; the nucleic acid hydrolysis liquid is 200U/L nucleic acid hydrolase;
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin with the antibiotic specific binding ssDNA, adding manganese ions with final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex;
the sequence of antibiotic specific binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 7
A time-resolved fluorescence immunochromatographic kit for rapidly detecting antibiotics, comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific ofloxacin binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, and is coated with ofloxacin antigen bovine serum albumin complex on a detection T line, and coated with goat anti-rabbit secondary antibody on a quality control C line; preparing a whole antigen with immunogenicity and reactivity from ofloxacin and bovine serum albumin, wherein the coupling ratio is 1: 100, and dialyzing and purifying after coupling to obtain an ofloxacin bovine serum albumin compound; diluting the ofloxacin bovine serum albumin compound 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 membrane spraying machine to prepare a detection T line; diluting the goat anti-rabbit secondary antibody to 1mg/mL by using PBS-sucrose buffer solution to prepare detection C line solution, and spraying the detection C line solution on a nitrocellulose membrane by using a membrane spraying machine to prepare a detection C line; drying the sprayed nitrocellulose membrane at a low temperature;
the cell lysis solution comprises the following components in percentage by mass: triton X-1003%, SDS 1% and NaCl 5%; the nucleic acid hydrolysis liquid is 200U/L nucleic acid hydrolase;
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor;
connecting olfactory protein and TYLCV-C1 protein through chemical modification to obtain connexin, connecting cgtataatatta sequence to the 5' end of the antibiotic specific binding ssDNA, connecting the connexin with the antibiotic specific binding ssDNA, adding manganese ions with final concentration of 6mM, and reacting at 25 ℃ for 10min to obtain a protein nucleic acid complex;
the sequence of antibiotic specific binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
example 8
The use method of the kit prepared by the invention comprises the following steps:
1) weighing 0.5g of tissue to be detected, placing the tissue to be detected in a homogenizing tube, adding 1mL of extracting solution into the homogenizing tube, and homogenizing to obtain homogenate;
2) placing the homogenate liquid on the upper end of the filter column, centrifuging to separate filter residue from filtrate, adding 1mL of filtrate to the upper end of the filter column again, centrifuging, and collecting the filtrate to obtain a liquid to be detected;
3) and (3) dripping the liquid to be detected into a sample pad, reading the fluorescence intensity of the T line and the fluorescence intensity of the 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 the prepared standard curve.
First, specific binding assay
Dissolving 1uL of specific ofloxacin-nucleic acid protein composite labeled by Eu fluorescent microsphere with concentration of 50umol/L into 198uL of PBS buffer solution, adding 1uL of oxyfloxacin of 10umol/L, uniformly mixing, adding protein precipitator to precipitate the specific ofloxacin-nucleic acid protein composite labeled by Eu fluorescent microsphere, centrifuging, collecting supernatant, measuring the content of oxyfloxacin in the supernatant by using conventional liquid chromatography, and repeating the test for 3 times, wherein the result shows that the content of oxyfloxacin in the supernatant is not detected. The Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein compound prepared by the invention can be specifically bound with the Ofloxacin.
Dissolving 100uL of specific ofloxacin combined nucleic acid protein compound marked by 50umol/L Eu fluorescent microsphere in PBS buffer solution of 19800uL, adding 100uL of ofloxacin/10 umol/L, uniformly mixing, adding protein precipitator to precipitate the specific ofloxacin combined nucleic acid protein compound marked by the Eu fluorescent microsphere, centrifuging, collecting supernatant, concentrating the supernatant to 20 times, measuring the content of ofloxacin in concentrated solution by using conventional liquid chromatography, and repeating the test for 3 times, wherein the content of ofloxacin in the concentrated solution is 0.13 ug/L.
Second, fluorescence enhancement test
1uL of Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein complex of 8mg/L is dissolved in 999uL of PBS buffer solution, and the emission spectrum is shown in figure 1 under the action of excitation wavelength of 270 nm.
1uL of Eu fluorescent microsphere of 8mg/L is dissolved in 999uL of PBS buffer solution, and the emission spectrum is shown in FIG. 1 under the action of excitation wavelength of 270 nm.
1uL of the connexin with the concentration of 8mg/L is dissolved in 999uL of PBS buffer, and the emission spectrum is shown in FIG. 1 under the effect of an excitation wavelength of 270 nm.
In fig. 1, 1 is a Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein complex solution; 2 is Eu fluorescent microsphere; and 3 is connexin.
The result shows that the fluorescence intensity of the specific ofloxacin-nucleic acid protein complex marked by the Eu fluorescent microsphere is obviously enhanced.
Thirdly, determining a quantitative standard curve
The Eu fluorescent microsphere labeled specific ofloxacin binding nucleic acid protein complex standard is respectively diluted into 0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.4ng/mL, 0.8ng/mL, 1.0ng/mL and 2.0ng/mL by PBS buffer solution, each concentration is detected for 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, a standard curve is obtained, and the result is shown in FIG. 2.
Fourthly, accuracy verification test
0.20ug/L of oxyflexacin solution is prepared, the concentration of oxyflexacin solution is detected by the kit (embodiment 7) and the liquid chromatogram respectively, the test is repeated for three times, the average detection concentration of the kit is 0.199ug/L, and the detection concentration of the liquid chromatogram is 0.21ug/L, which shows that the kit has higher detection accuracy.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. The invention is therefore not to be limited to the specific details described herein, without departing from the general concept as defined by the appended claims and their equivalents.
Claims (8)
1. A time-resolved fluorescence immunochromatography kit for rapidly detecting antibiotics is characterized by comprising:
the reagent strip comprises a plastic card shell, wherein a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad are arranged in an interlayer in the plastic card shell, the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially assembled on the PVC plate in a lap joint manner, the water absorption pad and the combination pad are respectively overlapped and pressed at two ends of the nitrocellulose membrane, the sample pad is overlapped and pressed on the combination pad, and a detection T line and a quality control C line are sequentially arranged on the nitrocellulose membrane;
the binding pad is adsorbed with specific fluoroquinolone antibiotic binding nucleic acid protein complex marked by Eu fluorescent microspheres and rabbit IgG antibody, a detection T line is coated with fluoroquinolone antibiotic antigen bovine serum protein complex, and a quality control C line is coated with goat anti-rabbit secondary antibody;
extracting solution, wherein the extracting solution comprises cell lysis solution and nucleic acid hydrolysate,
a filter column and a filtrate, the filtrate comprising a nucleic acid hydrolase inhibitor.
2. The time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics of claim 1, wherein the olfactory protein and TYLCV-C1 protein are linked by chemical modification or non-covalent bond to obtain a linker protein, the 5' end of the antibiotic-specific binding ssDNA is linked with cgtataatatta sequence, the linker protein is linked to the antibiotic-specific binding ssDNA, manganese ion is added to a final concentration of 6mM, and the reaction is carried out at 25 ℃ for 10min to obtain a protein nucleic acid complex.
3. The time-resolved fluoroimmunoassay kit for rapid detection of antibiotics of claim 1, wherein the sequence of the antibiotic specifically binding to ssDNA is:
5’-TAGGGAATTC 10
GTCGACGGAT 20
CCTGGCGCTT 30
AGGTGTAATA 40
ACCTGAGGAC 50
GGCTTGGCTG 60
CAGGTCGACG 70
CATGCGCCG-3’79。
4. the time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics of claim 1, wherein the fluoroquinolone antibiotics and bovine serum albumin are prepared into a whole antigen with immunogenicity and reactivity, the coupling ratio is 1: 100, and the fluoroquinolone antibiotics bovine serum albumin complex is obtained by dialysis purification after coupling.
5. The time-resolved fluorescence immunochromatographic kit for rapid detection of antibiotics of claim 1, wherein a fluoroquinolone antibiotic bovine serum albumin complex is diluted to 1mg/mL with PBS-sucrose buffer solution to prepare a detection T-line solution, and the detection T-line solution is spotted on a nitrocellulose membrane with a spotting machine to prepare a detection T-line; diluting the goat anti-rabbit secondary antibody to 1mg/mL by using PBS-sucrose buffer solution to prepare detection C line solution, and spraying the detection C line solution on a nitrocellulose membrane by using a membrane spraying machine to prepare a detection C line; drying the sprayed nitrocellulose membrane at low temperature.
6. The time-resolved fluoroimmunoassay kit for rapid detection of antibiotics of claim 1, wherein the cell lysate comprises the following components by mass fraction: triton X-1003%, SDS 1% and NaCl 5%; the nucleic acid hydrolysis liquid is 200U/L nucleic acid hydrolase.
7. The time-resolved fluoroimmunoassay kit for rapid detection of antibiotics according to any one of claims 1 to 6, wherein the kit is used in a method comprising the steps of:
1) weighing 0.5-1g of tissue to be detected, placing the tissue to be detected in a homogenizing tube, adding 1mL of extracting solution into the homogenizing tube, and homogenizing to obtain homogenate;
2) placing the homogenate liquid on the upper end of a filter column, centrifuging to separate filter residue from filtrate, adding 1mL of filtrate to the upper end of the filter column again, centrifuging, collecting filtrate, and mixing uniformly to obtain a liquid to be detected;
3) and (3) dripping the liquid to be detected into a sample pad, reading the fluorescence intensity of the T line and the fluorescence intensity of the C line by using a POTC analyzer after 5min, calculating a T/C value, and calculating the concentration of the fluoroquinolone antibiotics in the sample according to the prepared standard curve.
8. The time-resolved fluoroimmunoassay kit for rapid detection of an antibiotic according to claim 1, wherein the fluoroquinolone antibiotic is ofloxacin.
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| KR20140072480A (en) * | 2012-12-05 | 2014-06-13 | 아이알랩 주식회사 | development of a microbial inhibition test with indicator to detect residual fluoroquinolones in food |
| EP2774988A1 (en) * | 2013-03-08 | 2014-09-10 | Helmholtz-Zentrum für Umweltforschung GmbH-UFZ | Fluorchinolon-specific aptamers |
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