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Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications

A porcine blue-ear virus and detection test paper technology, which is applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of long maintenance time of N protein antibodies, achieve timely control of the epidemic, clear and easy to distinguish results, and save manpower and material resources.

Active Publication Date: 2012-11-14
辽宁迪浩生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the immune response stimulated by the N protein is the strongest, and the PRRSV antibody produced by the body is mainly directed against the N protein. After a pig is infected with PRRSV, the N protein antibody can be detected after one week, and the N protein antibody lasts for a long time

Method used

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  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications
  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications
  • Test paper strip for detecting PRRSV antibody colloidal gold, method for making same and applications

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Experimental program
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Effect test

Embodiment 1

[0047] Cloning and expression of embodiment 1 porcine blue ear virus N protein

[0048] (1) Obtaining the target gene

[0049] The genome of porcine PRRS virus was provided by Beijing Institute of Animal Husbandry and Veterinary Medicine. The pET-32a(+) expression vector and PMD-18T were purchased from Novagen. According to the characteristics of the pET-32a(+) expression vector, design primers with restriction endoenzymes BamHI and XhoI restriction sites at both ends:

[0050] Upstream is 5′C GGA TCC ATG CCA AAT AAC AAC G 3′

[0051] Downstream is 5'G CTC GAG TCA TGC TGA GGG TGA T 3'

[0052] The target fragment N was amplified, and the amplification conditions were: denaturation at 94°C for 3 minutes, 45s at 94°C, annealing at 56°C for 90s, extension at 72°C for 90s, 30 cycles, and finally extension at 72°C for 10 minutes.

[0053] (2) Cloning of the target gene and screening of positive recombinants

[0054] After electrophoresis, the PCR amplified product was recovere...

Embodiment 2

[0061] Embodiment 2: Preparation of polyclonal antibody against N protein of porcine blue ear virus

[0062] (1) Animal immunity:

[0063] New Zealand white rabbits of 1-2 kg were selected, and the N protein of porcine PRRS virus was injected subcutaneously at multiple points on the back, and the immunization dose was 0.5-1 mg / kg. A total of 3 to 5 times of immunization.

[0064] (2) Immunological titer detection:

[0065] Coat the porcine blue ear virus N protein ELISA plate, 4 μg per well. The titer of immune serum was detected by indirect ELISA method. When the serum titer reaches 1:20000 or more, the serum can be collected.

[0066] (3) Antibody purification and verification:

[0067] Purification by conventional octanoic acid method. The purity was tested by non-denaturing PAGE electrophoresis, showing a protein band. The activity was tested by ELISA, and the titer was greater than 1:16000.

Embodiment 3

[0068] Embodiment 3: the development of porcine blue ear virus antibody detection reagent strip (referring to Fig. 1)

[0069] (1) Preparation of colloidal gold-antigen conjugate:

[0070] It has been determined through experiments that the optimal pH value for the combination of PRRSV N protein and colloidal gold is 8.4, and the ratio of colloidal gold and PRRSV N protein is 36 μg / ml colloidal gold. After being treated with a stabilizer (0.5% BSA, pH 8.0, 0.01M Tris buffer), the labeled colloidal gold antigen was diluted to OD2.0, and the colloidal gold-antibody conjugate solution was taken at an amount of 65 μl per square centimeter, and evenly adsorbed On glass fiber, freeze-dried, and stored in a dry environment.

[0071] (2) Coating antigen and antibody on nitrocellulose membrane:

[0072] The N protein of porcine PRRS virus was diluted to 3.5±0.1mg / ml with 0.01MPBS. Dilute the polyclonal antibody against the N protein of porcine PRRS virus to 2±0.1mg / ml with 0.01MPBS....

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Abstract

The invention provides a test strip for rapid detection of a swine blue ear virus antibody. The swine blue ear virus antibody N protein, and anti swine blue ear virus antibody N protein polyclonal antibody are coated on a nitrate cellulose film (NC film), and a membrane chromatography double antigen sandwich method is adopted to detect the swine blue ear virus antibody in a swine serum, plasma, or whole blood specimen in combination with a colloidal gold labeled wine blue ear virus N protein. The test strip is simple in operation, convenient, and fast, and has the advantages of no requirements of special instruments and special training, clear and identified result, and easy popularization. The test strip is suitable for base course, large scale site detection of an accident and epidemiological investigation, and has auxiliary effect on the diagnosis of swine blue ear virus infection.

Description

technical field [0001] The invention relates to a colloidal gold detection test strip for porcine blue ear virus antibody, a preparation method and application thereof, and belongs to the field of biological detection. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), with high fever, high morbidity and mortality, adult pig reproductive disorders, premature birth, abortion and stillbirth, piglet respiratory Characterized by abnormalities, it is a contagious disease. The incidence of piglets can reach 100%, and the mortality rate is about 50%. The abortion and stillbirth of sows can reach more than 30%, which is very harmful to the pig industry. The disease occurs in almost all pig-raising countries in the world, and was introduced into my country in the mid-1990s. Because of its serious threat to the pig industry, the Int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/545
Inventor 刘明
Owner 辽宁迪浩生物科技有限公司
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