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Porcine circovirus 2-type antigen or antibody colloidal gold fast detection test strip and method

A porcine circovirus and colloidal gold technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of long operation time, complicated process, difficult to popularize and popularize at the grassroots level, etc., and achieve the effect of improving the recognition rate.

Inactive Publication Date: 2020-01-14
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods can detect the antigen and antibody of PCV2, but there are restrictions such as long operation time, complicated process, professional operators and special equipment, and it is difficult to popularize and promote at the grassroots level.

Method used

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  • Porcine circovirus 2-type antigen or antibody colloidal gold fast detection test strip and method
  • Porcine circovirus 2-type antigen or antibody colloidal gold fast detection test strip and method
  • Porcine circovirus 2-type antigen or antibody colloidal gold fast detection test strip and method

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Experimental program
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Effect test

Embodiment 1

[0064] The preparation of embodiment 1 porcine circovirus type 2 CAP antigen

[0065] Using the PCV2 (GU325755) sequence published by GenBank as a reference, the corresponding fragment was amplified, connected to the prokaryotic expression vector pET32a, and expressed by the prokaryotic expression system to obtain a soluble recombinant protein, namely porcine circovirus type 2 CAP recombinant protein. The corresponding gene sequence is shown in SEQ ID NO.5, which is used as the immune antigen of PCV2 after HIS affinity purification, and the protein purification result is as follows figure 1 shown.

Embodiment 2

[0066] Example 2 Preparation of anti-porcine circovirus type 2 CAP antigen polyclonal antibody

[0067] (1) Take 3 New Zealand white rabbits of 2.5Kg, the first dose is calculated based on the amount of 1 mg CAP recombinant protein per rabbit, add an equal volume of Freund's complete adjuvant for emulsification, and inject 6-8 points subcutaneously on the back, 0.1-0.2 points per point ml. Immunized every two weeks and then emulsified with Freund's incomplete adjuvant. Heart blood was collected 10 days after the third immunization to collect serum.

[0068] (2) Adjust the pH of the carbonate buffer solution of the recombinant CAP protein (10 mg) to 9.0, draw 1 ml of CNBr Sepharose FF and wash the solvent on the surface. Soak the packing material in the recombinant protein buffer, shake and mix at 25 degrees for 6 hours. Then add 0.1 mol / L Tris-HCl (pH 8.3) to continue the blocking treatment for 6 hours. Pack the filler into the column and wash 10 column bed volumes with 1m...

Embodiment 3

[0075] The mensuration of embodiment 3 antigen test strip detection sensitivity

[0076] The CAP recombinant protein was diluted with PBS, from 70μg / mL to 14μg / mL, 2.8μg / mL, 0.56μg / mL, 112ng / mL, 22.4ng / mL and 4.48ng / mL, each diluted The CAP recombinant protein and PBS as a control were dropped into the antigen detection test strip, and the results were as follows: Figure 4 As shown, the test strip can detect the lowest 22.4ng / mL CAP recombinant protein, and has good detection sensitivity.

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Abstract

The invention discloses a porcine circovirus 2-type antigen or antibody colloidal gold fast detection test strip and method. The test strip comprises a PVC plate, a sample pad, a gold standard pad, anitrocellulose membrane, a water absorption pad, a C-line and a T-line, wherein gold standard protein, namely CAP recombinant protein is adopted in the colloidal gold test strip, is dominant antigen epitope peptide of a denucleation positioning signal and is provided with an His label; the CAP recombinant protein is low-temperature induced and expressed to supernatant through a prokaryotic expression system; and anti-porcine circovirus 2-type CAP recombinant protein polyclonal rabbit serum marked on the T-line (antigen detection test strip) or the C-line (antibody detection test strip) is purified after being coupled to the CAP recombinant protein through CNBr activated agarose gel FF. Through the test strip, an antigen or antibody in porcine serum can be quickly detected, sensitivity is high, specificity is strong, good stability is achieved, operation is simple, different levels of demands can be well met, and popularization to a basic level is easy.

Description

technical field [0001] The invention belongs to a test strip of diagnostic technology in the field of biotechnology, in particular to a colloidal gold detection test strip for porcine circovirus type 2 (PCV2) antigen or antibody, a preparation method and a use method. Background technique [0002] Porcine circovirus 2 (PCV2) is the main pathogen that causes postweaning multisystemic wasting syndrome (PMWS) in piglets, causing progressive emaciation, slow growth, dyspnea and other symptoms in affected animals. In addition, PCV2 is also related to porcine dermatitis and nephrotic syndrome, proliferative necrotizing pneumonia, porcine respiratory disease syndrome, and reproductive disorders. Related diseases caused by PCV2 infection are widespread in the world, resulting in pig mortality ranging from 10% to 30%, causing severe economic losses to the pig industry. [0003] The main detection methods of porcine circovirus type 2 adopted at home and abroad include virus isolation...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569C07K14/01C12N15/34C12N15/70G01N33/536
CPCC07K14/005C12N15/70C12N2750/10022G01N33/536G01N33/56983G01N33/68G01N2333/01
Inventor 卓洵辉丁豪杰丁建祖陆绍红孔庆明陈睿郑斌童群波楼涤
Owner ZHEJIANG ACAD OF MEDICAL SCI
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