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

A poultry adenovirus and detection method technology, applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of unsuitable for large-scale detection of serum samples, the existence of loose poison, time-consuming, etc., to achieve the improvement of prokaryotic expression, The effect of short time consumption and simple operation

Active Publication Date: 2014-09-24
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 180 μL of allantoic fluid, add 20 μL of GA, 20 μL of proteinase K, mix well, and digest at 56°C for 4 hours.

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

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

[0049] (4...

Embodiment 2

[0134] Prepare 100K recombinant protein according to steps one to five in Example 1

[0135] 6. Establishment of 100K indirect ELISA detection method

[0136] The purified 100K recombinant protein was used as an antigen to establish an indirect ELISA method. The specific steps are as follows:

[0137] (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 100 μL per well to a 96-well microtiter plate, incubate at 37°C for 1 hour, and place at 4°C 12h, wash the plate 3 times with PBST, and pat dry;

[0138] (2) Blocking: add 200 μL of 5% skimmed milk to each well, block at 37°C for 1 hour, wash the plate three times with PBST, and pat dry;

[0139] (3) Binding with serum: add the serum sample to be tested, 100 μL / well, set positive and negative standard samples as controls, incubate at 37°C for 1 hour, wash the plate 3 times with PBST, and pat dry;

[0140] (4) Bind...

Embodiment 3

[0147] Prepare 100K recombinant protein according to steps one to five in Example 1

[0148] 6. Establishment of 100K indirect ELISA detection method

[0149] The purified 100K recombinant protein was used as an antigen to establish an indirect ELISA method. The specific steps are as follows:

[0150] (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 to 96-well ELISA plate, 100μL per well, incubate at 37°C for 1h, and place at 4°C 15h, wash the plate 3 times with PBST, and pat dry;

[0151] (2) Blocking: add 200 μL of 5% skimmed milk to each well, block at 37°C for 1 hour, wash the plate three times with PBST, and pat dry;

[0152] (3) Binding with serum: add the serum sample to be tested, 100 μL / well, set positive and negative standard samples as controls, incubate at 37°C for 1 hour, wash the plate 3 times with PBST, and pat dry;

[0153] (4) Binding to the enz...

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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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Patent Type & Authority Patents(China)
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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