Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold

A porcine circovirus and detection test paper technology, which is applied in the direction of analyzing materials through chemical reactions and material analysis through observing the impact on chemical indicators, can solve the problems of long detection time, low specificity, and stability. In order to achieve the effect of intuitive and easy to judge the test results, strong specificity and high safety

Active Publication Date: 2012-10-17
兆丰华生物科技(南京)有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing methods for detecting serum antibodies mainly include enzyme-linked immunosorbent assay (ELISA). In this traditional detection method, there are some shortcomings that are difficult to overcome. Longer; it needs to be used with a microplate reader in the detection, and the detection professionals also need to undergo professional training; the overall cost is high; the method also has the characteristics of low specificity and poor stability

Method used

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  • Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold
  • Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold
  • Test strip for quickly detecting porcine circovirus 2 (PCV2) antibody by adopting colloidal gold

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Expression and purification of embodiment 1PCV-2 nucleocapsid protein

[0027] (1) Extraction of PCV-2 ORF2 gene and amplification of target gene

[0028] The expression protein selected by this colloidal gold test strip is the nucleocapsid protein (Cap protein) of porcine circovirus type 2, and its corresponding gene is ORF2, so the following primers are designed:

[0029] Upstream primer: 5'-GATATCATGACGTATCAAAGCAGG-3'

[0030] Downstream primer: 5'-GTCGACCGTAGGGTTCAGAGGGGG-3'

[0031] Use the kit (Tiangen Biochemical Technology Co., Ltd.) to extract the DNA of PCV-2 virus as a template for the PCR reaction. The 50 μl reaction system of PCR consists of: 1 μl of PCR template, 1 μl of each primer (concentration is 50 mmol / L), 10×PCR buffer (containing MgCl 2 ) 5 μL, 4×dNTPs 4 μL (concentration is 2.5 mmol / L), Taq DNA polymerase 1 μL, dd H 2 O 38 μL. The reaction conditions of PCR were pre-denaturation at 94°C for 5min, denaturation at 94°C for 40s, annealing at 55°...

Embodiment 2

[0042] The preparation of embodiment 2 monoclonal antibody

[0043] (1) Animal immunity

[0044] Fully mix and emulsify the purified protein with Freund's complete adjuvant in equal volumes, and inject into the mouse intraperitoneally, the inoculation dose is 50 μg / mouse; 14 days later, inject the emulsion of the same purified protein and Freund's incomplete adjuvant into the mice After 28 days, inject the same purified protein and the emulsion of Freund's incomplete adjuvant into the peritoneal cavity of the mouse again; the last time the purified protein without adjuvant was boosted for immunization, and the spleen was aseptically taken three days later for fusion.

[0045] (2) Establishment of screening methods

[0046] Blood was collected from the immunized mice on the 21st day, 35th day and before spleen extraction, and the plasma was separated to obtain positive serum.

[0047] Use the purified recombinant protein as the coated antigen, dilute it with the coating solut...

Embodiment 3

[0062] The preparation of embodiment 3PCV-2 antibody colloidal gold test strips

[0063] (1) Colloidal gold labeled antigenic protein

[0064] Prepare gold solution by sodium citrate reduction method: add 2ml of 1.5% trisodium citrate solution to 50ml of boiling 0.04% chloroauric acid aqueous solution, continue stirring and heating until the solution is stable and bright red. Observing the colloidal gold solution under a transmission electron microscope, it is found that the colloidal gold particles are uniform in size, without oval or other irregular shapes, indicating that stable colloidal gold has been obtained.

[0065] With 0.1mol / L K 2 CO 3 Adjust the pH value of the colloidal gold between 7 and 10, and add the porcine circovirus type 2 Cap protein to be labeled to the adjusted pH value of the colloidal gold solution at a labeling ratio of 1:1000 to 2000, and let it stand for 10 minutes. Add 20% polyethylene glycol (PEG) 10000 to a final concentration of 0.05%, centri...

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Abstract

The invention discloses a test strip for quickly detecting a porcine circovirus 2 (PCV2) antibody by adopting colloidal gold. The test strip comprises a support layer, and a sample loading pad, a gold labelled antibody release pad, a detection layer and an absorption layer which are arranged on the support layer in sequence, wherein a colloidal gold labelled PCV2 nucleocapsid protein is embedded in the gold labelled antibody release pad; a detection strip and a quality control strip are arranged on the detection layer; a PCV2 nucleocapsid protein is immobilized on the detection strip; and a monoclonal antibody of the PCV2 nucleocapsid protein is immobilized on the quality control strip. The test strip has the following advantages: the test strip has strong specificity, the probability of false positive occurrence is greatly reduced, and the specificity and sensitivity of the test strip are ensured; the test strip has high safety and avoids operation of the virus in the detection process; the test strip is simple, convenient and quick to operate and does not need professional technicians for operation and professional instruments for auxiliary detection; and the detection result is intuitive and is easy to judge, and the detection time is only 10 minutes (the detection speed is extremely high).

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and relates to a rapid detection antibody technology, in particular to a detection test strip for porcine circovirus type 2 (PCV2) antibody, and also relates to a preparation method and an application method of the test strip. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) belongs to the family Circoviridae and the genus Circovirus. Among them, porcine circovirus type 2 (PCV2) is related to a variety of diseases in pigs, mainly causing postweaning multisystemic wasting syndrome (PMWS). After PCV2 infects pigs, it can lead to a decline in the immune function of pigs. This can easily lead to secondary or concurrent infections of other diseases. Therefore, porcine circovirus type 2 is a serious threat to the swine industry. [0003] Since 2001 in my country, there have been reports of PCV-2 infection cases all over the country. In order to better control porcine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78
Inventor 王贵华于倩倩王美君汤波陈义锋王洪彬赵亚荣
Owner 兆丰华生物科技(南京)有限公司
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