CN114544944A - Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant - Google Patents

Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant Download PDF

Info

Publication number
CN114544944A
CN114544944A CN202210048566.6A CN202210048566A CN114544944A CN 114544944 A CN114544944 A CN 114544944A CN 202210048566 A CN202210048566 A CN 202210048566A CN 114544944 A CN114544944 A CN 114544944A
Authority
CN
China
Prior art keywords
beta
stimulant
detection
pad
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210048566.6A
Other languages
Chinese (zh)
Inventor
闫立婷
王宗秀
付素姣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Yanqi Yueshengzhai Halal Food Co ltd
Original Assignee
Beijing Yanqi Yueshengzhai Halal Food Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Yanqi Yueshengzhai Halal Food Co ltd filed Critical Beijing Yanqi Yueshengzhai Halal Food Co ltd
Priority to CN202210048566.6A priority Critical patent/CN114544944A/en
Publication of CN114544944A publication Critical patent/CN114544944A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/58—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
    • G01N33/587—Nanoparticles
    • 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
    • G01N33/9406—Neurotransmitters
    • 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
    • G01N33/9406—Neurotransmitters
    • G01N33/9413—Dopamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nanotechnology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention provides a detection card for detecting livestock and poultry beta-stimulant and a method for detecting the beta-stimulant. The provided detection card comprises a sample pad, a combination pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially adhered on the bottom plate, a colloidal gold-labeled antibody is contained on the combination pad, and the colloidal gold-labeled antibody has HCDR sequences shown in SEQ ID NO 1, 2 and 3 and LCDR sequences shown in SEQ ID NO 4, 5 and 6; the reaction membrane is a nitrocellulose membrane, a detection line (T) and a quality control line (C) are arranged on the nitrocellulose membrane, the detection line is coated with a beta-stimulant drug hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse anti-antibody. The application of the detection card can realize the low detection limit detection of the beta-stimulant.

Description

Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to a detection card for detecting livestock and poultry beta-stimulant and a method for detecting the beta-stimulant.
Background
The harm of beta-stimulant to human is very big, mainly expressed in: palpitation, tremor of muscles of face, neck and limbs, tremor of fingers, feeling of heavy sensation in feet or even incapability of standing, headache, dizziness, nausea, vomiting, hypodynamia, flushing of face, allergic red pimple of skin appear in acute poisoning. The symptoms of hypertension, coronary heart disease and hyperthyroidism are more likely to occur, and the harm to mammals is similar to the symptoms. Sudden death may even occur when the tolerance is weak and stressors are present for a long time. The livestock and poultry fed with the beta-stimulant often have the condition of acute heart failure and sudden death in long-distance transportation. For example, clenbuterol has stable properties and will decompose only when heated to 172 ℃, so that the structure of clenbuterol cannot be destroyed by ordinary cooking methods at all. Studies have shown that if taken for a long period of time, it may cause chromosome aberration, which may induce malignant tumors.
The beta-stimulant can be detected by adopting an integrated rapid detection card. The method has the characteristics of convenience, rapidness and sensitivity in industrial production, is suitable for detecting a large amount of industrialized samples, and improves the industrialization. However, further improvements are needed for the detection of this method.
Disclosure of Invention
The invention aims to provide a method for integrally detecting beta-stimulant, which can comprehensively and objectively detect most stimulant components. Meanwhile, the detection is carried out on livestock and poultry such as pigs, cattle, sheep and the like, but not on a single livestock and poultry.
Specifically, the invention provides the following technical scheme:
the invention provides a detection card for detecting livestock and poultry beta-stimulant, which comprises the following components:
the sample pad, the combination pad, the reaction membrane, the water absorption pad and the bottom plate are sequentially stuck on the bottom plate,
the binding pad contains a colloidal gold-labeled antibody, and the colloidal gold-labeled antibody has HCDR sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and LCDR sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
the reaction membrane is a nitrocellulose membrane, and a detection line (T) and a quality control line (C) are arranged on the nitrocellulose membrane, wherein the detection line is coated with a beta-stimulant drug hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse anti-antibody.
Further, the beta-stimulant drug hapten-carrier protein conjugate is obtained by coupling beta-stimulant drug hapten and carrier protein.
Further, the carrier protein is selected from at least one of bovine serum albumin, ovalbumin and human serum albumin.
In a second aspect of the present invention, there is provided a method for detecting β -agonists in livestock and poultry using a detection card according to the first aspect of the present invention, the method comprising:
a step of applying a sample to the sample-absorbing pad of the test card according to the first aspect of the present invention to carry out the test.
Further, when the content of the substance to be detected in the sample is higher than the detection limit, the test area is not colored, and the result is positive;
when the content of the substance to be detected in the sample is lower than the detection limit or the beta-stimulant is not contained, the test area is colored, and the result is negative.
Further, the method has a detection limit of 3ng/g (3ppb) for clenbuterol (Cle);
the method has a detection limit of 3ng/g (3ppb) for ractopamine (Ract);
the method has a detection limit for salbutamol (Sal) of 5ng/g (5 ppb).
The beneficial effects obtained by the invention are as follows:
the invention is suitable for industrial mass detection, and has the advantages of simple and rapid operation, low cost and high accuracy. And the detection limit requirement of the sample is stricter than the national standard. For example, detection limit: clenbuterol (Cle): 3ng/g (3 ppb); ractopamine (Ract): 3ng/g (3 ppb); salbutamol (Sal):5ng/g (5 ppb).
Drawings
Fig. 1 is a schematic structural diagram of a detection card for detecting β -agonists in livestock and poultry according to an embodiment of the present invention, in which reference numeral 1 is a sample pad, 2 is a binding pad, 3 is a reaction membrane, 4 is a water absorption pad, 5 is a bottom plate, 6 is a detection line, and 7 is a quality control line.
Detailed Description
The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention. Some of the terms herein are explained and illustrated, which are also used to assist those skilled in the art in understanding.
The invention provides a detection card for detecting livestock and poultry beta-stimulant, which comprises the following components: the kit comprises a sample pad, a combination pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially adhered on the bottom plate, the combination pad contains a colloidal gold labeled antibody, and the colloidal gold labeled antibody has HCDR sequences shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 and LCDR sequences shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6; the reaction membrane is a nitrocellulose membrane, and a detection line (T) and a quality control line (C) are arranged on the nitrocellulose membrane, wherein the detection line is coated with a beta-stimulant drug hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse anti-antibody.
The colloidal gold-labeled antibody mentioned herein can be obtained by labeling the antibody by a method commonly used in the art. The mentioned goat anti-mouse anti-antibodies can be obtained commercially. Reagents not mentioned herein are either commercially available or available through common general knowledge of those skilled in the art.
Example 1
Firstly, a conjugate of a hapten of a beta-stimulant drug and a carrier protein is used as an immunogen to immunize a mouse, so as to obtain an antibody capable of being specifically combined with the beta-stimulant drug. Selected from among antibodies exhibiting high affinity, having HCDR sequences represented by GFSPSGYG (SEQ ID NO:1, HCDR1), INDEDYA (SEQ ID NO:2, HCDR2), GRYYYAADI (SEQ ID NO:3, HCDR3) and LCDR sequences represented by ESYYTRNR (SEQ ID NO:4, LCDR1), RAS (SEQ ID NO:5, LCDR2), AGRGSRTTDIA (SEQ ID NO:6, LCDR3) were subjected to sequencing analysis.
Example 2
Embodiment 2 provides a detection card, as shown in fig. 1, including: the device comprises a sample pad 1, a combination pad 2, a reaction membrane 3, a water absorption pad 4 and a bottom plate 5, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially adhered on the bottom plate; the binding pad contains a colloidal gold labeled antibody, and the colloidal gold labeled antibody has HCDR sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and LCDR sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; the reaction membrane is a nitrocellulose membrane, the nitrocellulose membrane is provided with a detection line (T, shown as 6 in figure 1) and a quality control line (C, shown as 7 in figure 1), the detection line is coated with a beta-stimulant drug hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse anti-antibody.
When the content of the substance to be detected in the sample is higher than the detection limit, the detection line (T line) does not develop color, and the result is positive; on the contrary, when the sample does not contain the to-be-detected object or the content of the to-be-detected object is lower than the detection limit, the detection line (T line) is colored, the result is visible to naked eyes, and the result is negative.
Example 3
Example 3 livestock and poultry beta-agonists were tested using the test card provided in example 2.
First, a sample is pretreated
1. Before the sample is processed, it is necessary to know: the laboratory instruments must be cleaned and disposable tips used to avoid contamination from interfering with the results of the experiment.
2. A sample pretreatment step:
organizing: weighing 5g +/-0.05 g of homogeneous fresh muscle tissue sample, putting the muscle tissue sample into a 50ml centrifuge tube with a cover, and carrying out boiling water bath for 10 min; centrifuging for 1 minute at 4000 rpm, cooling, and taking supernatant for detection.
The specific operation steps are as follows:
1. taking out the detection card, unsealing, placing on a table, carefully sucking the supernatant of the centrifuge tube by using a dropper, and adding 3 drops of the supernatant into the sample hole.
2. And 5min after sample addition, observing a color development area, and judging a result.
Result judgment
1. Negative: the quality control line (C line) is colored, the detection line (T line) is visible by naked eyes, and the color is judged to be negative no matter the color is dark or light; indicating that the sample does not contain corresponding clenbuterol or the content of the clenbuterol is lower than the detection limit;
2. positive: the quality control line (C line) is colored, and the detection line (T line) is not colored, so that the result is positive; indicating that the corresponding clenbuterol content in the sample is higher than the detection limit;
3. and (4) invalidation: the quality control line (C line) and the detection line (T line) do not develop color or the quality control line (C line) does not develop color but the detection line (T line) develops color; the test card is invalid, overdue, or improperly operated, and another test is required.
The detection is carried out on 100 samples, and the result shows that the detection limit of the method for clenbuterol (Cle) is 3ng/g (3 ppb); the method has a detection limit of 3ng/g (3ppb) for ractopamine (Ract); the detection limit of the method for salbutamol (Sal) is 5ng/g (5ppb), and the accuracy rate is more than 99%.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims (6)

1. A detection card for detecting livestock and poultry beta-stimulant is characterized by comprising:
the device comprises a sample pad, a combination pad, a reaction membrane, a water absorption pad and a bottom plate, wherein the sample pad, the combination pad, the reaction membrane and the water absorption pad are sequentially adhered to the bottom plate;
the binding pad contains a colloidal gold-labeled antibody, and the colloidal gold-labeled antibody has HCDR sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and LCDR sequences shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;
the reaction membrane is a nitrocellulose membrane, a detection line (T) and a quality control line (C) are arranged on the nitrocellulose membrane, the detection line is coated with a beta-stimulant drug hapten-carrier protein conjugate, and the quality control line is coated with a goat anti-mouse anti-antibody.
2. The test card of claim 1, wherein the β -agonist drug hapten-carrier protein conjugate is obtained by conjugating a β -agonist drug hapten to a carrier protein.
3. The test card of claim 2, wherein the carrier protein is at least one selected from the group consisting of bovine serum albumin, ovalbumin, and human serum albumin.
4. A method for detecting livestock and poultry beta-stimulant by using the detection card of any one of claims 1-3, which is characterized by comprising the following steps:
a step of adding a sample to the sample pad of the test card according to any one of claims 1 to 3 for testing.
5. The method of claim 4, wherein when the content of the substance to be tested in the sample is higher than the detection limit, the test area is not colored and the result is positive;
when the content of the substance to be detected in the sample is lower than the detection limit or the beta-stimulant is not contained, the test area is colored, and the result is negative.
6. The method of claim 4, wherein the method has a detection limit for clenbuterol (Cle) of 3ng/g (3 ppb);
the method has a detection limit of 3ng/g (3ppb) for ractopamine (Ract);
the method has a detection limit for salbutamol (Sal) of 5ng/g (5 ppb).
CN202210048566.6A 2022-01-17 2022-01-17 Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant Pending CN114544944A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210048566.6A CN114544944A (en) 2022-01-17 2022-01-17 Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210048566.6A CN114544944A (en) 2022-01-17 2022-01-17 Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant

Publications (1)

Publication Number Publication Date
CN114544944A true CN114544944A (en) 2022-05-27

Family

ID=81672523

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210048566.6A Pending CN114544944A (en) 2022-01-17 2022-01-17 Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant

Country Status (1)

Country Link
CN (1) CN114544944A (en)

Similar Documents

Publication Publication Date Title
US8058011B2 (en) Method for the measurement of endocrine substances in an analyte
CN109709339B (en) Colloidal gold immunochromatographic test strip for detecting skeletal muscle troponin I of cattle or sheep and application thereof
CN112964873B (en) SARS-CoV-2 detecting reagent kit based on sandwich method
DK174032B1 (en) Kit as well as immunometric dosing method that can be applied to whole cells
WO2016064545A1 (en) Lateral flow immunoassay methods and devices for simultaneously detecting hemoglobin s, hemoglobin c, and hemoglobin a in newborns, infants, children, and adults
CN204989196U (en) Qualitative test paper strip of immunity chromatography is united to six antinuclear antibodiess
CN201087838Y (en) Myocardium calcium protein I color particle diagnosis test paper
CN105334323A (en) Method and test strip for detecting zilpaterol, and application of test strip
US4842995A (en) Diagnostic method for the evaluation of clinical parameters by direct collection of biological materials and device for its accomplishment
JPH09512101A (en) Method for detecting the presence of Mycobacterium species and kits and antibodies used in the method
CN112964874A (en) SARS-CoV-2 detecting reagent kit based on indirect method
CN114544944A (en) Detection card for detecting livestock and poultry beta-stimulant and method for detecting beta-stimulant
CN106483300A (en) A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application
Ahmad et al. A review Hair tissue analysis: an analytical method for determining essential elements, toxic elements, hormones and drug use and abuse
CN203178274U (en) Novel clenbuterol multi-residue colloidal gold test card
JPH05113443A (en) Enzyme-immunity measuring method
CN111141907A (en) Florfenicol rapid detection kit and detection method
JP3709078B2 (en) Method and kit for measuring diacetylpolyamine
Leventhal et al. Cuticular reactivity of early larval stages of Ascaris suum: binding of fractions of immune serum to the surface of infective and parasitic stage larvae as detected by the mixed antiglobulin test
CN114675022A (en) Detection card for detecting chloramphenicol in beef and mutton and method for detecting chloramphenicol
CN109541232A (en) A kind of detection anti-HAV kit and preparation method thereof
LU505995B1 (en) Label-free surface plasmon resonance immunosensor for rapid determination of salbutamol residue in animal urine
JPS5961777A (en) Analytical system of bile acid
CN202256342U (en) Rapid detection card for detecting beta agonist residues in animal blood
JPH05346428A (en) Kit for rapidly counting granulocyte and method using the kit

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination