Magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody

A technology of fluorescence immunity and swine fever virus, which is applied in the field of animal disease detection, can solve the problems that it is not suitable for widespread use by grass-roots veterinary departments, cannot realize on-site detection of samples, increases detection time and difficulty, and achieves long fluorescence decay time and overcomes difficulties. Effects of stability and shortening of reaction time

Inactive Publication Date: 2018-06-12
GUANGZHOU BIOKEY HEALTH TECH CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) The forward indirect hemagglutination test is recommended by the Ministry of Agriculture as the main method for the detection of swine fever vaccine antibodies, but its sensitivity is low, and the detection serum needs to be inactivated, which increases the detection time and difficulty, and the error of naked eye interpretation is large, so it is not suitable for the grassroots Widely used in the veterinary sector
[0005] (2) Competitive ELISA method: Although it has strong specificity and high accuracy, the operation process is cumbersome and requires professional technicians
[0006] (3) Colloidal gold / fluorescence immunochromatography: Although the operation is simple and the detection time is short, the accuracy is often not high, the quantification cannot be accurate, and the detection range is narrow
[0007] (4) Chemiluminescence method: Although it has the advantages of high sensitivity and wide detection range, it requires professional equipment and technical personnel, and cannot realize on-site detection of samples
[0008] (5) RT-PCR: It has the advantages of high detection rate and high specificity, but requires expensive instruments and equipment, and the detection time is long (4-6 hours). Improper operation is prone to false positive and false negative results

Method used

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  • Magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody
  • Magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody
  • Magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 preparation method:

[0035] A detection kit for swine fever virus antibody, comprising: reagent strips, negative control serum and positive control serum.

[0036] The reagent strip is mainly coated with immunomagnetic beads of classical swine fever virus antigen E2 protein, europium-labeled sheep anti-pig antibody, sample diluent, washing solution, and enhancing solution;

[0037] (1) Preparation method of classical swine fever virus antigen E2 protein:

[0038] ① Amplification of CSFV E2 protein

[0039] A pair of primers were designed according to the gene sequence of the attenuated CSFV strain published on GenBank (GenBank accession number is AF531433) for amplifying the E2 gene.

[0040] Primers P1: 5'-CGCGGATCCCGGCTAGCCTGC-3'; P2: 5'-CCGCTCGAGTTATAGTACCTGTTCT-3'.

[0041] ②Construction of recombinant plasmid pEGFP-E2

[0042] The amplified classical swine fever virus E2 gene was connected to the eukaryotic expression vector pEGFP-C1 vector by clo...

Embodiment 2

[0065] Embodiment 2 This kit usage method:

[0066] ① Add sample

[0067] Put the sample to be tested into the loading system of the automatic fluorescence analyzer, insert the reagent strip into the reagent strip slot, and the instrument will automatically identify the product information of the sealing film. Add 20 μL of the sample to the 8th well, dilute 32 times, then take 50 μL of the diluted sample and add it to the 1st well of the reagent strip, and then add 100 μL of the fluorescent marker.

[0068] ② Incubation

[0069] After adding samples, shake and incubate at 37°C for 15 minutes.

[0070] ③ washing

[0071] After the incubation is completed, the instrument automatically washes the wells 5 times, each washing solution is 100 μL / well.

[0072] ④Add enhancement solution

[0073] After washing, 150 μL / well of enhancement solution was added and incubated at 37°C for 3 minutes.

[0074] ⑤ Detection

[0075] The reagent strip is pushed into the dark room, and the ...

Embodiment 3

[0079] Embodiment 3 The specificity experiment of this kit

[0080] Magnetic Particle Fluorescent Immunoassay Kit for Quantitative Detection of Antibody to CSFV in Specific Experiments to Detect CSFV (CSFV), Bovine Viral Diarrhea Virus (BVDV), Sheep Border Virus (BDV), Porcine Pseudorabies (PRV) and Pig Blue Ear (PRRSV), porcine parvovirus (PPV) and other standard positive serum and CSF negative serum, except CSFV standard serum is 95840, the fluorescence value of the rest of the serum is less than 20022, in line with the criteria for negative serum, indicating that this method is specific Good, and there is no cross-reaction with BVDV and BDV positive sera, the test results are shown in Table 1.

[0081] Table 1 Specificity experiments

[0082] virus type

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Abstract

The invention discloses a magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody, which consists of classical swine fever E2 antigen immunomagnetic beads, fluorescent substance-labeled goat anti-pig antibody, detergent, enhancement solution, negative control serum and positive control serum. Compared with the RT-PCR (reverse transcription-polymerase chain reaction) method and the ELISA (enzyme-linked immunosorbent assay) method, the magnetic micro particle immunofluorescence kit for quantitatively assaying classical swine fever virus antibody which is disclosed by the invention is easy to operate and suitable for assaying different numbers of samples, and has high assay speed (a result can be obtained in about 20 minutes); and compared with the colloidal gold immunochromatographic strip, the kit has the advantages of higher sensitivity, wider linear range, capability of realizing accurate quantification and the like. The assay result of the kit can accurately reflect the change law of anti-CSFV (anti-classical swine fever virus) serum IgG antibody of the body in the process of vaccination and provide scientific and reasonabletechnical support for pig farmers in preventing and controlling CSF (classical swine fever).

Description

technical field [0001] The invention belongs to the field of animal epidemic detection, in particular to a magnetic particle fluorescence immunoassay kit for quantitative detection of swine fever virus antibody. It is suitable for detecting the level of swine fever virus antibody in pig serum or plasma. Background technique [0002] Classical swine fever (CSF) is an acute, febrile, contagious disease of pigs caused by classical swine fever virus (CSFV). The World Health Organization classifies it as a Class A infectious disease. The disease spreads quickly, is popular, has a high incidence rate and a high lethality rate, and is extremely harmful to the pig industry in my country. At present, the prevention and control measures of swine fever in my country are mainly based on the immunization of the attenuated swine fever rabbit vaccine, and the evaluation of the immune effect of the vaccine is mainly realized by the detection of the antibody level of the swine fever virus....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/531
CPCG01N33/6857G01N33/531G01N33/5432
Inventor 李根平
Owner GUANGZHOU BIOKEY HEALTH TECH CO LTD
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