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ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection

An avian adenovirus and a detection method technology, which are applied to measurement devices, biological tests, material inspection products, etc., can solve the problems of being unsuitable for the detection of large-scale serum samples, the existence of scattered poison, time-consuming and other problems, and achieve the improvement of prokaryotic expression, The effect of short time and simple operation

Active Publication Date: 2012-09-19
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are time-consuming and laborious, and are not suitable for the detection of large quantities of serum samples, and most of the antigens used are whole viruses, and there is a risk of loose virus
However, there is no research report on the ELISA detection method using 100K recombinant protein to identify group I adenovirus infection

Method used

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  • ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection
  • ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection
  • ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Cloning of 100K genes

[0044] 1. Extraction of group I poultry adenovirus DNA

[0045] Use the Tiangen Biochemical Technology Blood / Cell / Tissue Genomic DNA Extraction Kit to extract DNA directly from the allantoic fluid of chicken embryo lethal orphan virus (CELOV) seed poison passage purchased from the China Veterinary Drug Administration, and operate according to the kit Manual, the specific steps are as follows:

[0046] (1) Take 180uL of allantoic fluid, add 20uLGA, 20uL proteinase K, mix well, and digest at 56°C for 4h.

[0047] (2) Add 200uL buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0048] (3) Add 200uL of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent sediment may appear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0049] (4) Add the solution and...

Embodiment 2

[0151] Prepare 100K recombinant protein according to steps one to five in Example 1

[0152] 6. Establishment of 100K indirect ELISA detection method

[0153] The purified 100K recombinant protein was used as an antigen to establish an indirect ELISA method. The specific steps are as follows:

[0154] (1) Coating: Dilute the 100K protein of group I poultry adenovirus expressed by the prokaryotic to the working concentration of 1ug / mL with the coating solution, add to a 96-well ELISA plate, 100uL per well, incubate at 37°C for 1h, and place at 4°C 12h, wash the plate 3 times with PBST, and pat dry;

[0155] (2) Sealing: add 200uL 5% skimmed milk to each well, seal at 37°C for 1 hour, wash the plate 3 times with PBST, and pat dry;

[0156] (3) Binding with serum: add the serum sample to be tested, 100uL / well, set positive and negative standard samples as controls, incubate at 37°C for 1h, wash the plate 3 times with PBST, and pat dry;

[0157] (4) Binding to enzyme-labeled se...

Embodiment 3

[0164] Prepare 100K recombinant protein according to steps one to five in Example 1

[0165] 6. Establishment of 100K indirect ELISA detection method

[0166] The purified 100K recombinant protein was used as an antigen to establish an indirect ELISA method. The specific steps are as follows:

[0167] (1) Coating: Dilute the 100K protein of group I poultry adenovirus expressed by prokaryotic to the working concentration of 10ug / mL with the coating solution, add 100uL per well to a 96-well ELISA plate, incubate at 37°C for 1h, and place at 4°C 15h, wash the plate 3 times with PBST, and pat dry;

[0168] (2) Sealing: add 200uL 5% skimmed milk to each well, seal at 37°C for 1 hour, wash the plate 3 times with PBST, and pat dry;

[0169] (3) Binding with serum: add the serum sample to be tested, 100uL / well, set positive and negative standard samples as controls, incubate at 37°C for 1h, wash the plate 3 times with PBST, and pat dry;

[0170] (4) Binding to the enzyme-labeled se...

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Abstract

The invention discloses an ELISA (enzyme-linked immunosorbent assay) detection method for identifying fowl adenovirus group I (FAVI) infection. The method is characterized by taking an FAVI 100K recombinant protein as the envelop antigen, chicken serum as the detection sample and horse radish peroxidase labeled goat anti-chicken IgG as the enzyme-labeled antibody to detect the antibody generated through FAVI infection. The method is strong in specificity, short in time and low in cost, is simple to operate, can be used for mass detection, can effectively get rid of interference of immunity of FAVI inactivated vaccines and can specifically detect FAVI infection, thus distinguishing FAVI infected animals from FAVI inactivated vaccine inoculated animals and providing a diagnostic tool with actual value for eliminating FAVI in China.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and particularly relates to an indirect ELISA method for using 100K recombinant protein to distinguish animals infected by group I adenovirus and animals immunized with inactivated vaccine of group I avian adenovirus. Background technique [0002] Fowl adenovirus group I (Fowl adenovirus group I, hereinafter referred to as FAVI) belongs to the genus of adenoviridae and has a common group antigen. Based on the hexon gene sequence, it is divided into 5 different species (A-E), based on the neutralization test As a result, it was subdivided into 12 serotypes. FAVI is ubiquitous in the world, and its hosts are mostly in chickens, ducks, and geese, and can be isolated from healthy and diseased poultry. The disease can be transmitted both horizontally through excrement and vertically through eggs, contaminating chicken embryos. In recent years, diseases such as inclusion body hepatitis, ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/531
Inventor 谢芝勋罗思思刘加波邓显文庞耀珊谢志勤谢丽基范晴彭宜
Owner GUANGXI VETERINARY RES INST
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