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Indirect ELISA kit for detecting aviadenovirus group I antibody, and detection method and application thereof

A poultry adenovirus and kit technology, applied in measuring devices, color/spectral characteristic measurement, analysis through chemical reaction of materials, etc., can solve the problems of inaccurate results, time-consuming and labor-consuming, etc., and achieve low cost and reliable technology Means, effects that are easily identified and controlled

Inactive Publication Date: 2017-08-29
YANGLING VOCATIONAL & TECHN COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the traditional laboratory diagnostic methods for FAV Ⅰ have certain defects, such as virus isolation and identification, neutralization test, agar diffusion test and other methods are time-consuming and labor-intensive, and the results are not accurate enough. PCR, LAMP, etc. have high requirements for reagents and equipment. It is inconvenient to detect a large number of samples, but ELISA has the advantages of simplicity, rapidity, and strong specificity, and ELISA detection of antibodies can effectively evaluate the immune effect of vaccines

Method used

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  • Indirect ELISA kit for detecting aviadenovirus group I antibody, and detection method and application thereof
  • Indirect ELISA kit for detecting aviadenovirus group I antibody, and detection method and application thereof
  • Indirect ELISA kit for detecting aviadenovirus group I antibody, and detection method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Construction of FAV Ⅰ Hexon protein (Hexon refers to hexon protein) expression vector:

[0035] Genomic DNA of FAV Ⅰ CELO strain (CVCC AV208) (purchased from China Veterinary Drug Administration) was extracted, and the synthetic primer HexonF:5′-CGC was designed GGATCC ATGACTGCGCTTACTCCCGA-3' (the underline is the BamHI restriction site), HexonR:5'-CCG CTCGAG CCGCTCGAGCGCCAGCATGTACTGGTAAC-3' (the underline is the XhoI restriction site), amplified to obtain the Hexon gene amplification product, the length of the amplified product is 1017bp, its nucleotide sequence is detailed in SEQ.ID.No.1, gel recovery Hexon gene purpose The fragment (amplified product) was ligated to the cloning vector pEASY-T1, and ligated overnight at 16°C. The ligated product was transformed into Trans5α clone competent cells, and spread onto a kanamycin-resistant (100mg / ml) LB plate, and placed at 37°C Cultivate overnight, pick a single clone, shake culture in kanamycin-resistant LB medium f...

Embodiment 2

[0063] Specific test of ELISA kit:

[0064]According to the established indirect ELISA operation method, use the ELISA kit to detect the known positive serum samples of Group I poultry adenovirus, Newcastle disease, avian influenza, Salmonella, Staphylococcus aureus, Pasteurella multocida and Escherichia coli respectively; Measure the OD value of each well with a microplate reader at a wavelength of 450nm to determine the specificity of the indirect ELISA detection method;

[0065] The test results showed that only the OD of FAV Ⅰ positive serum 450 >0.322, other serum OD 450 Between 0.112-0.201, far less than 0.322, indicating that the specificity of the indirect ELISA test result is good.

Embodiment 3

[0067] Repeated tests within the same batch of ELISA kits:

[0068] Use the FAV Ⅰ Hexon recombinant protein prepared by induction and purification in the same batch, coat the microtiter plate according to the optimal coating concentration, and use the established indirect ELISA operation method to detect 3 positive sera and 3 FAV Ⅰ negative sera with different FAV Ⅰ antibody levels. The 6 randomly selected sera were detected at different times, and the OD value was measured with a microplate reader at a wavelength of 450nm; according to the OD value of each well 450 Value, calculate the average value of each serum Standard deviation S, coefficient of variation CV; test results showed that 6 serum OD 450 The coefficient of variation of the value is between 1.2%-3.5%, indicating that the same batch of FAV Ⅰ Hexon recombinant protein detection results have good repeatability.

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Abstract

The invention discloses an indirect ELISA kit for detecting an aviadenovirus group I antibody, and a detection method and an application thereof and relates to the technical field of biological detection. The ELISA kit disclosed by the invention comprises a coated elisa plate, a washing liquid, a diluting liquid, a primer developing liquid, a rabbit-anti-chicken elisa second antibody, a terminating liquid, FAVI standard positive serum and FAVI standard negative serum, wherein the coated elisa plate takes a recombinant hexon protein of aviadenovirus group I FAVI as a coating antigen. The ELISA kit disclosed by the invention is used for detecting the aviadenovirus group I antibody, has the advantages of high efficiency, sensitive specificity and repeatability, and is simple and fast to operate, low in cost and suitable for clinical application and popularization.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an indirect ELISA kit for detecting group I poultry adenovirus antibodies, a detection method and application thereof. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAV) is a non-enveloped double-stranded DNA virus, which can be divided into three groups: I, II, and III. Group I fowl adenovirus (fowl adenovirus group I, FAV I) has a common group The representative strain of this group of viruses is Chicken Embro Lethal Orphan Virus (CELOV). Since June 2015, Angrara disease (Angrara disease) has broken out in chicken flocks in most provinces of my country. Characterized by hepatitis, pericardial effusion syndrome, and gizzard erosion. The disease mainly occurs in 3-week-old broilers. The peak of death is about 1 week after the onset, and the mortality rate can reach 20% to 80%, causing huge economic losses to breeding and production. At present, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577G01N33/543G01N33/535G01N21/78G01N21/31
CPCG01N33/56983G01N21/31G01N21/78G01N33/535G01N33/543G01N33/577G01N2333/075
Inventor 张曼杨书会
Owner YANGLING VOCATIONAL & TECHN COLLEGE
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