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Colloidal gold detection kit for detecting avian leukosis virus and application thereof

The technology of an avian leukosis virus and a detection kit is applied in the field of avian leukosis virus detection, which can solve the problems of being unable to be popularized and applied on a large scale in grass-roots farms, long detection time, complicated operation, etc., and achieves low cost, short time consumption, and simple operation. Effect

Active Publication Date: 2019-03-26
CHINA ANIMAL DISEASE CONTROL CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The first object of the embodiments of the present invention is to provide a colloidal gold detection kit for detecting avian leukosis virus, which is used to solve the problem of high sensitivity of the colloidal gold detection kit in the prior art and the long detection time of the existing ELISA technology. The operation is relatively complicated and requires a microplate reader, which can only be tested in laboratories or central laboratories, and cannot be widely applied in grassroots farms.

Method used

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  • Colloidal gold detection kit for detecting avian leukosis virus and application thereof
  • Colloidal gold detection kit for detecting avian leukosis virus and application thereof
  • Colloidal gold detection kit for detecting avian leukosis virus and application thereof

Examples

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Embodiment 1

[0044] This embodiment is a preparation method of avian leukosis virus P27 protein monoclonal antibody, the preparation method comprising the following steps:

[0045] One, the preparation of avian leukosis virus P27 protein:

[0046] According to the sequence design primer of GenBank sequence number JF911742.1, construct carrier, transfer into Escherichia coli BL21 (DE3) and construct recombinant engineering bacterium; The engineering bacterium (designated as pET-28a(+)-P27- BL21(DE3)) was inoculated in LB liquid medium containing ampicillin 100 μg / ml, and activated overnight at 37°C; subsequently, the activated culture was inoculated into 5 ml of LB liquid medium containing ampicillin 100 μg / ml at a volume ratio of 3% In the medium, cultivate at 37°C until the OD600nm reaches 0.7, add isopropylthiogalactoside, and make the final concentration of isopropylthiogalactoside 0.2mmol / L, at 37°C at 250r / min After 4 hours of induction, collect the bacterial solution and transfer it...

Embodiment 2

[0061] The present embodiment is the preparation method of the specific monoclonal antibody 5F1 of colloidal gold-labeled anti-fowl leukosis virus p27 protein, and the preparation method comprises the following steps:

[0062] (1) Select 10ml of colloidal gold solution with a mass concentration of 1.5% into a centrifuge tube, and add 50 μl of 0.1M K 2 CO 3 solution;

[0063] (2) Add 20 μl of 1 mg / ml monoclonal antibody 5F1 solution against avian leukosis virus p27 protein to the colloidal gold solution, and incubate at room temperature for 20 minutes; then, add bovine serum albumin to the incubated solution to mix well, and make The concentration of bovine serum albumin is 0.2% (w / v), and the precipitate is collected by centrifugation for 40 min at 10000 r / min;

[0064] (3) Redissolve the precipitate with 0.02M phosphate buffer containing 1% sucrose, 1% trehalose, 2% BSA, and 0.02% sodium azide to obtain the specificity of the colloidal gold-labeled anti-avian leukosis virus...

Embodiment 3

[0066] The present embodiment is the preparation method of microporous reagent, and this preparation method comprises the following steps:

[0067] (1) Dilute the colloidal gold-labeled specific monoclonal antibody 5F1 against avian leukosis virus p27 protein to 5 μg / mL with 0.05 mol / L pH9.6 carbonate buffer solution, and then dilute the diluted solution to 100 μL per well Add to 96-well ELISA plate and coat at 6°C for 16h;

[0068] (2) Discard the coating solution in the microplate wells, add 300 μL of PBS-Tween solution with a concentration of 0.1% pH 7.2 to each well of the microplate to wash the plate 3 times, each time for 3 minutes, then pat dry, and then Add 200 μL of 1.0% bovine serum albumin to the wells of the microtiter plate, and block at 37°C for 2 hours;

[0069] (3) Discard the bovine serum albumin solution in the microplate well, add 250 μ L concentration to each hole of the microplate and be the PBS-Tween solution of 0.1% pH 7.2 to wash the plate for 3 minute...

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Abstract

The embodiment of the invention discloses a colloidal gold detection kit for detecting avian leukosis virus and application thereof. The colloidal gold detection kit comprises a detection test strip and a micropore reagent, wherein the detection test strip comprises a nitrocellulose membrane; the nitrocellulose membrane is provided with a detection line and a quality control line; the detection line is coated by a monoclonal antibody 1A1 resisting p27 protein of avian leukosis virus; the micropore reagent is a micropore containing the colloidal gold-marked monoclonal antibody 5F1 resisting thep27 protein of the avian leukosis virus. The colloidal gold detection kit has the advantages that the sensitivity in detection is high, the consumption time is short, and the detection efficiency ishigh; the operation is simple, the difficulty in operation is low, the cost is low, and the colloidal gold detection kit is suitable for being popularized and applied to the detection laboratory at base level.

Description

technical field [0001] The invention relates to the technical field of avian leukosis virus detection, in particular to a colloidal gold detection kit for detecting avian leukosis virus and its application. Background technique [0002] Avian Leukosis Disease (Avian Leukosis Disease) is a viral infectious disease caused by Avian Leukosis Virus (Avian Leukosis Virus, ALV). Cellular leukemia, followed by erythroblastic leukemia and myeloblastic leukemia. In addition, it can also cause myeloid cell tumors, connective tissue tumors, epithelial tumors, endothelial tumors, etc. Most tumors invade the hematopoietic system, and a few invade other tissues. [0003] Avian leukosis virus ALV belongs to the genus Alpharetrovirus in the family Retroviridae. According to the antigenicity of its envelope protein, ALV has been divided into ten subgroups A-J so far. ALV mainly causes infection and disease in chicken flocks. It can not only cause subclinical pathogenic effects such as imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558G01N33/577
CPCG01N33/558G01N33/56983G01N33/577
Inventor 顾小雪赵荣茂王传彬杨春江蒋菲吴佳兴杨林莫勋宋晓晖孙海霞刘玉良韩雪
Owner CHINA ANIMAL DISEASE CONTROL CENT
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