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Enzyme-linked immuno sorbent assay (ELISA) kit for detecting sheep pseudo rabies virus (PRV) antibody

A pseudorabies virus and kit technology, which is applied in the field of indirect ELISA kits to achieve the effects of high sensitivity, good stability and strong specificity

Active Publication Date: 2014-12-03
SHANDONG LVDU BIO SICIENCE & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many commercial detection kits available at home and abroad, but most of the detection objects are for the most common and susceptible pigs

Method used

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  • Enzyme-linked immuno sorbent assay (ELISA) kit for detecting sheep pseudo rabies virus (PRV) antibody
  • Enzyme-linked immuno sorbent assay (ELISA) kit for detecting sheep pseudo rabies virus (PRV) antibody
  • Enzyme-linked immuno sorbent assay (ELISA) kit for detecting sheep pseudo rabies virus (PRV) antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Expression and purification of recombinant gD protein

[0045] 1) Primer design and synthesis

[0046] According to the gD gene sequence of the PRV Fa strain with the accession number AY196984 published by GenBank, a pair of gD gene primers were designed using Oligo6.0 and Primer software, and EcoRI and XhoI restriction sites were respectively introduced to amplify a fragment of 1059 bp. The primer sequences are as follows :

[0047] P1: 5'-GGG GAATTC TACCCGTACAC-3'

[0048] P2: 5'-GGG CTCGAG CCTCAGGCGGA-3'

[0049] 2) Extraction of PRV genomic DNA

[0050] Take 200 μl of PRV Fa strain (purchased from China Veterinary Microbiology Culture Collection Management Center) virus solution, and use a small amount of virus DNA / RNA extraction kit (purchased from AXYGEN, USA) to extract viral DNA.

[0051] 3) PCR amplification and cloning of PRV gD gene

[0052] Using the extracted PRV Fa strain DNA as a template, add the designed specific primers to PCR ampli...

Embodiment 2

[0059] Example 2: Preparation of antigen-coated plates

[0060] (1) gD diluted in coating buffer is loaded on a microwell plate

[0061] Specifically, the coating method includes 0.05M pH 9.6 carbonic acid buffer as the coating solution, adding 0.1 ml of the coating solution containing gD to each well of the microwell plate, and coating at 4° C. overnight for 16 hours.

[0062] (2) washing plate

[0063] Discard the liquid in each well into the waste liquid bucket, and add 250 μl of washing solution to each well (the 20× concentrated washing solution in the kit must be diluted with distilled water or deionized water before use. If there is crystallization, it must be dissolved before use. for dilution) to wash the plate, repeat 2 times, 30s each time; finally pat dry the microwell plate.

[0064] (3) closed

[0065] Add 0.13ml of blocking solution to each well of the microwell plate, place at room temperature for 2.5 hours, shake off the blocking solution in the well, and f...

Embodiment 3

[0070] Embodiment 3: the preparation of positive serum and negative serum

[0071] 1) Positive serum preparation

[0072] Select healthy goats with a body weight of 15-20kg, and inject 5.0mL of inactivated pseudorabies vaccine (purchased from Shandong Lvdu Biotechnology Co., Ltd.) subcutaneously in the neck. The second immunization is carried out 28 days after the first immunization, and the dose is the same as before; the second immunization After 14 days, blood was collected under sterile conditions to separate serum, and serum neutralization test (refer to OIE Terrestrial Animal Diagnostic Test and Vaccine Manual) was used to determine neutralizing antibodies; A large amount of blood was collected and serum was prepared at -20°C for future use.

[0073] 2) Negative serum preparation

[0074] Select healthy goats with a body weight of 15-20kg that have not been vaccinated and have not been infected with pseudorabies. Under sterile conditions, blood is collected to separate...

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Abstract

The invention relates to the technical field of animal epidemic disease diagnosis, and in particular relates to an enzyme-linked immuno sorbent assay (ELISA) kit for detecting a sheep pseudo rabies virus (PRV) antibody. The ELISA kit comprises an envelop plate, a positive control, a negative control, sample diluent, an enzyme label conjugate, 20*concentration washing liquid, substrate liquid, stop buffer, sealing glue, a self-sealing bag and an operation instruction; the kit is capable of rapidly, sensitively and accurately detecting the PRV antibody in the body of a sheep, so that the blank in the field at home and abroad is made up for. The kit can be used for replacing traditional serological methods such as a virus neutralization test, so that the high-flux and large-scale detection on the PRV antibody in the body of the sheep, with the high sensitivity, strong specificity and good stability, can be realized.

Description

technical field [0001] The invention relates to the technical field of animal disease diagnosis, in particular to an indirect ELISA kit for detecting pseudorabies virus antibodies in sheep, so as to monitor the levels of pseudorabies virus antibodies in sheep flocks and the distribution and prevalence of diseases. Background technique [0002] Pseudorabies, also known as Ojezki's disease, or PRV for short, is caused by herpes A virus of the herpesviridae family. The virus can infect the central nervous system and other organs, such as the respiratory tract, of all mammals except humans and apes. The disease can harm sheep at various stages, with a mortality rate as high as 100%. Clinically, it is characterized by fever, severe itching and symptoms of encephalomyelitis. The disease occurs and is widespread in the world. In recent years, with the development of my country's mutton sheep industry and the frequent flow of sheep, the disease has brought huge economic losses ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/54373G01N33/54393G01N33/56994G01N2333/032
Inventor 王金良沈志强董艳凯董炳梅陈金龙魏凤
Owner SHANDONG LVDU BIO SICIENCE & TECH
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