African swine fever virus detection test paper, kit and preparation method thereof

Abstract

The invention provides African swine fever virus test paper, a kit and a preparation method of the test paper. The African swine fever virus test paper comprises a back plate; a sample pad, a latex microsphere pad, a nitrocellulose membrane and a water absorption pad are sequentially arranged on the back plate; the latex microsphere pad is coated with a labeled antibody labeled by latex microspheres; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a capture antibody, and the detection line is arranged close to one sideof the latex microsphere pad; the quality control line is coated with a goat-anti-mouse anti-antibody, and the quality control line is arranged close to one side of the water absorption pad; and thelabeled antibody is a monoclonal antibody secreted by a hybridoma cell 7A7 with a preservation number of CGMCC No.18540 or a monoclonal antibody secreted by a hybridoma cell 3E5 with a preservation number of CGMCC No.18539, and the capture antibody is an African swine fever virus antibody. The African swine fever virus test paper can realize rapid, high-specificity and high-sensitivity detection of the African swine fever virus.

Description

Technical field

[0001] The invention belongs to the technical field of animal disease detection, and specifically relates to an African swine fever virus detection test paper, a kit and a preparation method thereof. Background technique

[0002] African swine fever (ASF) is a hemorrhagic, high-fatality, viral infectious disease of pigs caused by African swine fever virus (ASFV). Pigs of all ages are susceptible to infection. The incubation period of ASF natural infection is long, and the natural infection is 4-19 days. The average death time after infection is 2-10 days. The clinical manifestations include high fever, loss of appetite, skin and internal organ bleeding, etc. The clinical symptoms are similar to swine fever and swine Danqing virus. After pigs or wild boars are infected with African swine fever virus, the incubation period is usually 3-15 days, and the mortality rate of virulent strains can reach 100%. The World Organization for Animal Health (OIE) believes that i...

Examples

Embodiment 1

[0055] Example 1 Preparation of monoclonal antibody against p72 recombinant protein of African swine fever virus

[0056] 1. African swine fever virus p72 recombinant protein gene cloning and recombinant expression vector construction

[0057] According to the African swine fever virus p72 gene sequence (sequence number: AAT84439.1) published by Genbank as a reference, the optimized gene sequence was designed to make the N / C-terminal epitope of the antigen more easily exposed, showing more advantages. Therefore, a more suitable monoclonal antibody is screened. The present invention analyzes the dominant epitope region of the protein sequence through DNAstar and IEDB databases, and uses GGGGS as the linker to express the two main epitope regions in tandem, and the two epitopes The amino acid sequences of the regions are SEQ ID No. 1 and SEQ ID No. 2, which retain more amino acids while minimizing the impact of steric hindrance. The optimized nucleotide sequence of the African swine...

Embodiment 2

[0098] Example 2 Preparation of monoclonal antibody against African swine fever virus p72 recombinant protein labeled with latex microspheres

[0099] The prepared African swine fever virus p72 recombinant protein monoclonal antibody (7A7) is diluted with the diluent and labeled with latex microsphere particles. The specific operation is as follows:

[0100] 1. Cleaning of latex microspheres: Measure a certain volume of latex microspheres with a particle size of 300nm (purchased from Suzhou Weidu Biotechnology Co., Ltd., item number DR05C), pour it into a clean centrifuge tube, and add each 100 μL latex microspheres Add the labeling buffer to the ratio of 900 μL labeling buffer (50 mM MES, pH 6.0). Centrifuge at 17000 r / min for 10 min, remove the supernatant, add 1000 μL of labeling buffer, centrifuge at 17000 r / min for 10 min, remove the supernatant, and add 1000 μL of labeling buffer to resuspend the microspheres.

[0101] 2. Activation of latex microspheres: Weigh 20 mg of NHS an...

Embodiment 3

[0104] Example 3 Preparation of latex microsphere pad

[0105] 1. Take out the latex microsphere labeled African swine fever virus p72 recombinant protein monoclonal antibody solution from the refrigerator at 4°C and return to room temperature.

[0106] 2. Take a piece of 20cm×30cm glass fiber and cut it into 0.6cm×30 cm size with an accessory cutting machine.

[0107] 3. Spread a layer of cling film on the table, place the cut glass fiber on the cling film, and use a pipette to suck 1 mL of the latex microsphere labeled African swine fever virus p72 recombinant protein monoclonal antibody solution to evenly moisten it A cut glass fiber. Dry at room temperature for 12 to 16 hours, then transfer to a 40°C oven for 1 hour, then place in a sealed bag with desiccant, seal, and set aside.

Technical field

[0001] The invention belongs to the technical field of animal disease detection, and specifically relates to an African swine fever virus detection test paper, a kit and a preparation method thereof. Background technique

[0002] African swine fever (ASF) is a hemorrhagic, high-fatality, viral infectious disease of pigs caused by African swine fever virus (ASFV). Pigs of all ages are susceptible to infection. The incubation period of ASF natural infection is long, and the natural infection is 4-19 days. The average death time after infection is 2-10 days. The clinical manifestations include high fever, loss of appetite, skin and internal organ bleeding, etc. The clinical symptoms are similar to swine fever and swine Danqing virus. After pigs or wild boars are infected with African swine fever virus, the incubation period is usually 3-15 days, and the mortality rate of virulent strains can reach 100%. The World Organization for Animal Health (OIE) believes that i...

Examples

Embodiment 1

[0055] Example 1 Preparation of monoclonal antibody against p72 recombinant protein of African swine fever virus

[0056] 1. African swine fever virus p72 recombinant protein gene cloning and recombinant expression vector construction

[0057] According to the African swine fever virus p72 gene sequence (sequence number: AAT84439.1) published by Genbank as a reference, the optimized gene sequence was designed to make the N / C-terminal epitope of the antigen more easily exposed, showing more advantages. Therefore, a more suitable monoclonal antibody is screened. The present invention analyzes the dominant epitope region of the protein sequence through DNAstar and IEDB databases, and uses GGGGS as the linker to express the two main epitope regions in tandem, and the two epitopes The amino acid sequences of the regions are SEQ ID No. 1 and SEQ ID No. 2, which retain more amino acids while minimizing the impact of steric hindrance. The optimized nucleotide sequence of the African swine...

Embodiment 2

[0098] Example 2 Preparation of monoclonal antibody against African swine fever virus p72 recombinant protein labeled with latex microspheres

[0099] The prepared African swine fever virus p72 recombinant protein monoclonal antibody (7A7) is diluted with the diluent and labeled with latex microsphere particles. The specific operation is as follows:

[0100] 1. Cleaning of latex microspheres: Measure a certain volume of latex microspheres with a particle size of 300nm (purchased from Suzhou Weidu Biotechnology Co., Ltd., item number DR05C), pour it into a clean centrifuge tube, and add each 100 μL latex microspheres Add the labeling buffer to the ratio of 900 μL labeling buffer (50 mM MES, pH 6.0). Centrifuge at 17000 r / min for 10 min, remove the supernatant, add 1000 μL of labeling buffer, centrifuge at 17000 r / min for 10 min, remove the supernatant, and add 1000 μL of labeling buffer to resuspend the microspheres.

[0101] 2. Activation of latex microspheres: Weigh 20 mg of NHS an...

Embodiment 3

[0104] Example 3 Preparation of latex microsphere pad

[0105] 1. Take out the latex microsphere labeled African swine fever virus p72 recombinant protein monoclonal antibody solution from the refrigerator at 4°C and return to room temperature.

[0106] 2. Take a piece of 20cm×30cm glass fiber and cut it into 0.6cm×30 cm size with an accessory cutting machine.

[0107] 3. Spread a layer of cling film on the table, place the cut glass fiber on the cling film, and use a pipette to suck 1 mL of the latex microsphere labeled African swine fever virus p72 recombinant protein monoclonal antibody solution to evenly moisten it A cut glass fiber. Dry at room temperature for 12 to 16 hours, then transfer to a 40°C oven for 1 hour, then place in a sealed bag with desiccant, seal, and set aside.

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