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Colloidal gold testing paper card used for detecting rabies virus antibodies of dogs and cats, preparation method and application thereof

A technology of colloidal gold test paper and rabies virus, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems that the same test paper card cannot be tested, requires instruments, and complicated operation, etc., so as to reduce the detection procedures and have a long storage period Effect

Active Publication Date: 2010-10-06
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It overcomes the shortcomings of the existing detection technology, such as complex operation, cumbersome operation, long detection time, the need for instruments, and the inability to use the same test paper card to detect antibodies in different animals

Method used

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  • Colloidal gold testing paper card used for detecting rabies virus antibodies of dogs and cats, preparation method and application thereof
  • Colloidal gold testing paper card used for detecting rabies virus antibodies of dogs and cats, preparation method and application thereof
  • Colloidal gold testing paper card used for detecting rabies virus antibodies of dogs and cats, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of colloidal gold

[0038] Take a pickled and silicified 250mL Erlenmeyer flask, add 1mL of 1% chloroauric acid and 99mL of pure water, heat to boiling; accurately and quickly add 1.5mL of freshly prepared and filtered 1% trisodium citrate while stirring, in Within 3 minutes, the solution in the Erlenmeyer flask turned from light yellow to gray-black, then purple-black, and finally purple-red. Continue heating and stirring for 15 minutes, cool to room temperature, add pure water to restore the original volume, and the solution is a colloidal gold solution. Store in a refrigerator at 4°C, protected from light, for later use.

Embodiment 2

[0039] Embodiment 2: Cloning and expression of rabies virus N gene

[0040] ① Cloning of the target gene

[0041] According to the characteristics of the pET-32a(+) expression vector, a pair of primers containing restriction endonuclease EcoR I and Xho I restriction sites on both sides were designed, and the upstream primer RV NF: 5'-CTGC GAATTC ACGATGGATGCCGACAAGATTGT-3'; downstream primer RV NR: 5'-GCCA CTCGAG TTATGAGTCACTCGAATACGT-3'.

[0042] The total RNA in RV Flury LEP venom was extracted by Trizol method, and cDNA was synthesized, which was used as template for PCR amplification of RV N gene. After the PCR product was recovered by the agarose gel recovery kit, it was ligated with the pGM-T vector, transformed into E.coli DH5α competent cells, cultured overnight at 37°C, and the plasmid was extracted, and the correct recombinant plasmid was identified by enzyme digestion and sequencing. Named pGM-N.

[0043] ② Construction of N gene fusion expression vector

[00...

Embodiment 3

[0049] Embodiment 3: the preparation of the colloidal gold test paper card of detection dog of the present invention, cat rabies virus antibody

[0050] (1) Colloidal gold-labeled SPA and its purification

[0051] Get the colloidal gold solution that embodiment 1 makes, with 0.1mol / L K 2 CO 3 Adjust the pH to 5.3, slowly add an appropriate amount of 1mg / mL SPA dropwise under a magnetic stirrer to make the final concentration 3μgSPA / mL colloidal gold, continue stirring for 15min, and place at room temperature for 20min. Slowly add an appropriate amount of 3% (w / v) polyethylene glycol 20000 (PEG-20000) to make the final concentration 0.3% (w / v), continue stirring for 15 minutes, and place at room temperature for 15 minutes. Then centrifuge the solution at 3000r / min at 4°C for 10min, suck out the supernatant, and discard the black precipitate; then centrifuge the supernatant at 12000r / min at 4°C for 15min; discard the supernatant and keep the precipitate, and restore the precip...

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Abstract

The invention relates to a colloidal gold testing paper card used for detecting rabies virus antibodies of dogs and cats. By simultaneously coating a rabies virus-associated antigen strip and a staphylococcal protein A (SPA) antibody strip on a nitrocellulose membrane, and labeling the SPA by adopting colloidal gold, a universal testing paper card is obtained. The rabies virus antibodies in serums of the dogs and the cats are detected by using an immunochromatography principle so as to fulfill an aim of detecting the rabies virus antibodies of the dogs and the cats. The testing paper card overcomes the shortages of complicated and cumbersome operation, long testing time, requirement on instruments and incapability of detecting the antibodies in different animals by using the same testing paper card in the prior detecting art.

Description

technical field [0001] The invention relates to a colloidal gold test paper card for detecting rabies virus antibodies in dogs and cats, belonging to the field of pet antibody detection. Background technique [0002] Rabies is a zoonotic disease caused by rabies virus (RV). So far, there is no effective method to treat rabies, once the onset, the mortality rate is almost 100%. According to the World Health Organization, 50,000 to 100,000 people worldwide die from rabies every year. Dogs and cats are the main source of infection for rabies. Therefore, the key to the control of rabies is to cut off the source of infection and immunize dogs and cats. However, whether the immunity of dogs and cats is effective requires testing the antibody levels in their bodies. Currently, commonly used methods for detecting RV antibodies include the traditional mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT) recommended by WHO, and the fluorescent antib...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/531
Inventor 李刚范晓娟张坤
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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