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Colloidal gold immunochromatographic test paper detecting African swine fever virus antigen, preparation method and application thereof

An immunochromatographic test paper and African swine fever virus technology, which is applied in the field of virus epidemic diagnosis technology and animal quarantine, can solve the problems of difficult preparation of recombinant antigens, further verification of virus strain detection results, and virus spread.

Active Publication Date: 2019-01-22
ROHI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, ELISA is an international trade inspection method stipulated by OIE, but the detection antigen needs to be prepared from virus-infected cells, virus culture has the risk of spreading the virus, and the diagnostic reliability needs to be improved. The results need to be verified by methods such as Western-blotting
In order to solve these problems, recombinant antigen ELISA attempts are being made at home and abroad. Commercial kits have been supplied in France and Spain. However, due to the difficulty in preparing recombinant antigens, not only the supply of kits is limited and the price is expensive, but also the virus strains in different regions The test results need to be further verified

Method used

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  • Colloidal gold immunochromatographic test paper detecting African swine fever virus antigen, preparation method and application thereof
  • Colloidal gold immunochromatographic test paper detecting African swine fever virus antigen, preparation method and application thereof
  • Colloidal gold immunochromatographic test paper detecting African swine fever virus antigen, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 detects ASFV antigen colloidal gold immunochromatography test paper preparation

[0079] 1. Preparation of p30 recombinant antigen

[0080] The artificially synthesized and codon-optimized ASFV p30 gene (SEQ ID No.1) was inserted into the plasmid pET30a(+) to construct the recombinant plasmid pET-p30, and transform the competent cell BLR. According to the volume ratio of 1:100, the recombinant bacteria carrying plasmid pET-p30 were inoculated into 2×YT medium containing 50 μg / mL kanamycin, and cultured at 37°C until OD 600 = 0.8, add 1mmol / LIPTG, induce at 37°C for 4h; centrifuge at 8000rpm for 2min, wash the bacteria obtained by centrifugation twice with 0.01M PBS solution, and centrifuge again; the bacteria obtained by centrifugation again are lysed with an ultrasonic instrument, the power of the ultrasonic instrument is 50W, 10 s / time, 10 min in total; centrifuge at 4°C, 12,000 rpm for 20 min, and collect the precipitate; purify p30 recombinant protein ...

Embodiment 2

[0112] Embodiment 2 colloidal gold immunochromatography test paper minimum detection sensitivity determination

[0113] Dilute the purified recombinant protein p30 to 943 μg / mL, 9.43 μg / mL, 94.3 ng / mL, and 0.943 ng / mL, draw 85 μL of the dilution respectively, add it to the sample hole of the test paper, and observe the results within 5 minutes. The results showed that when the samples of four different concentrations were detected, red bands could appear on the quality control line and the detection line, while the color of the detection line was slightly lighter when the sample with a concentration of 0.943ng / mL was detected ( Figure 4 ), show that this colloidal gold immunochromatography test paper can detect the minimum ASFV p30 protein of 80pg.

Embodiment 3

[0114] Example 3 Colloidal gold immunochromatographic test paper detects ASFV p30 antigen in pig blood cells

[0115] 1. Transfection of PK15 cells with pcDNA-p30 plasmid

[0116] 3 μg pcDNA-p30 eukaryotic expression plasmid and 9 μL Lipofectamine2000 (Invitrogen) were dissolved in 500 μL Opti-MEM medium (Gibco) respectively, and allowed to stand at room temperature for 20 min. After mixing the two, continue to stand at room temperature for 5 min; the 6-well cell culture plate ( Corning) to 95% of the PK15 cell culture medium was discarded, washed with sterile PBS, added the above transfection mixture, 37 ° C 5% CO 2 Let it stand in the incubator for 4 hours; discard the transfection mixture and replace it with DMEM medium (Gibco) containing 1% fetal bovine serum. After 48 hours, trypsinize the PK15 cells, centrifuge the cells, suspend them in sterilized PBS, and set up pcDNA3. 0 empty vector transfection control group.

[0117] Take a small amount of suspended cells, add an...

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Abstract

The invention relates to colloidal gold immunochromatographic test paper detecting African swine fever virus antigen, a preparation method and application thereof, belonging to virus disease diagnosistechnologies and the field of animal quarantine. The test paper comprises a PVC bottom board, wherein a sample pad, a colloidal gold pad, a nitrocellulose membrane and a water absorption pad are fixed on the PVC bottom board in sequence, the colloidal gold pad is internally provided with one epitope of monoclonal antibody Mab1 of an anti-ASFV p30 antigen marked by colloidal gold, the surface of the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is designed for another epitope of monoclonal antibody Mab2 for ASFVp30 antigen, and the quality control line is a goat anti-mouse IgG antibody. The colloidal gold immunochromatographic test paper has the advantages of being convenient, rapid, sensitive and specific, can detect ASFV infectedcells directly, and is suitable for ASFV infected diagnosis, epidemiologic investigation and international trade quarantine for swine.

Description

technical field [0001] The invention belongs to the field of virus epidemic diagnosis technology and animal quarantine, and in particular relates to a colloidal gold immunochromatographic test paper for detecting African swine fever virus antigen, a preparation method and application thereof. Background technique [0002] African swine fever (ASF) is a highly contagious infectious disease of pigs. The highly pathogenic ASFV infection of domestic pigs can lead to 100% mortality. The disease has been endemic in many African countries and has now spread to Gruzi Neighboring countries such as Asia, Armenia, Azerbaijan and Russia pose a huge threat to my country's pig industry. Because there is no vaccine for ASF epidemic prevention at present, fast and accurate diagnosis is very important to prevent the spread and epidemic of the disease. The ASF diagnostic techniques recommended by the World Organization for Animal Health (OIE) include pathogen detection, nucleic acid identific...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/569
CPCG01N33/56983G01N33/577G01N33/6893
Inventor 张鑫宇刘潇羽孙怀昌周花艳
Owner ROHI BIOTECH
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