Universal indirect ELISA kit for detecting type 1 and type 3 duck hepatitis A virus serum antibodies, and application thereof

A technology for duck hepatitis A and serum antibodies, which is applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of long detection cycle, complicated operation, double cost, etc., and achieves reduced detection cost, good specificity, and simple operation. Effect

Active Publication Date: 2019-08-06
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the serological detection method for duck hepatitis A mainly adopts serum neutralization test, but this method has the disadvantages of long detection period and poor repeatability, and is not suitable for rapid detection. Reliable Detection Technology for Duck Hepatitis Antibody
The ELISA method established based on the whole virus is difficult to purify DHV, which limits the popularization and application of this method. The ELISA method established by the main antigenic protein expressed by E. In the evaluation and clinical mixed infection serum antibody detection, it is necessary to detect DHAV-1 and DHAV-3 serum antibodies separately, which is cumbersome and costs double
At present, there are few reports on the universal ELISA detection method for the serum antibody of duck hepatitis A virus type 1 and type 3 DHAV mainly prevalent in my country

Method used

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  • Universal indirect ELISA kit for detecting type 1 and type 3 duck hepatitis A virus serum antibodies, and application thereof
  • Universal indirect ELISA kit for detecting type 1 and type 3 duck hepatitis A virus serum antibodies, and application thereof
  • Universal indirect ELISA kit for detecting type 1 and type 3 duck hepatitis A virus serum antibodies, and application thereof

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Embodiment 1

[0059] Example 1 Establishment of Indirect ELISA Detection Method for Type 1 and Type 3 Duck Hepatitis A Virus Serum Antibodies

[0060] 1. Amplification of DHAV1-VP0 and DHAV3-VPO genes and construction of expression vectors

[0061] 1) Design DHAV-1-VP0 and DHAV-3 with reference to the complete gene sequence of type 1 duck hepatitis A virus E53 strain (EF151313.1) published by GenBank and the complete gene sequence of type 3 duck hepatitis A virus cloned in our laboratory - Two pairs of primers for VPO, DHAV-1-VP0 upstream primer: 5'- CCGGAATTCATGGATACTTCTCACCAAAAA-3'; DHAV-1-VP0 downstream primer: 5'- CCGCTCGAGTAACTGATTGTCAAATGGTCG-3'. DHAV-3-VP0 upstream primer: 5'-GGCGAATTCATGGACACTCTAACTA-3'; DHAV-3-VPO downstream primer: 5'-CGGCTCGAGTTACTGGTCATTGAAAGGCCG-3'. Using the positive plasmid carrying the DHAV-1VPO gene or DHAV-3VPO gene stored in our laboratory as a template, the target gene was amplified by PCR. Use the primers in Table 1 and Prime STAR DNA polymerase for P...

Embodiment 2

[0098] The assembly and application of embodiment 2 kit

[0099] 1. Kit assembly

[0100] The kit includes: type 1 DHAV VPO recombinant protein-coated ELISA plate, sample diluent, 10× concentrated washing solution, enzyme conjugate working solution, chromogenic solution A, chromogenic solution B, stop solution, positive serum and negative serum. Wherein, the sample diluent is 0.05M pH7.4 phosphate buffer containing 0.05% v / v Tween-20; the 10× concentrated washing solution is 0.01 M pH7.4 phosphate buffer; the enzyme conjugate working solution is HRP-goat anti-duck IgG; the chromogenic solution A is 0.2mg / mL of tetramethylbenzidine solution, the chromogenic solution B It is a citric acid-phosphate buffer solution containing 0.5‰w / v hydrogen peroxide urea; the stop solution is 0.01M sulfuric acid solution; the positive serum is duck hepatitis A virus type 1 vaccine and type 3 virus allantois The positive serum obtained from adult ducks immunized with liquid; the negative seru...

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Abstract

The invention discloses a universal indirect ELISA kit for detecting type 1 and type 3 duck hepatitis A virus serum antibodies, and an application thereof. The invention establishes a universal indirect ELISA detecting method for detecting type 1 and type 3 DHAV serum antibodies based on VP0 recombinant proteins. The method takes prokaryotically expressed type 1 DHAV VP0 recombinant proteins as detection antigens, and verifies that the VP0 recombinant proteins can specifically react with type 1 DHAV serum antibodies and can specifically react with type 3 DHAV serum antibodies by adopting Western blotting and indirect ELSIA methods, thus whether detected duck serum contains antibodies of type 1 and type 3 duck hepatitis A viruses and its antibody level can be determined by conducting indirect ELISA detection through taking the type 1 DHAV VP0 recombinant proteins as the detection antigens. The universal indirect ELISA kit and its application provided by the invention provide new and rapid detection means for effectively preventing and treating duck hepatitis A.

Description

technical field [0001] The invention particularly relates to a universal indirect ELISA kit for detecting serum antibodies of type 1 and type 3 duck hepatitis A virus based on the VPO protein of type 1 duck hepatitis A virus and its application. The invention belongs to the technical field of biological detection. Background technique [0002] Duck Viral Hepatitis (DVH) is a rapidly spreading and highly lethal virus in ducklings caused by Duck Hepatitis A Virus (DHAV) and Duck Astrovirus (DAstV) disease. Duck hepatitis A virus is a member of the genus Avian Hepadnavirus in the family Picornaviridae, and its members are divided into A, B, and C types according to their genotypes, respectively corresponding to the traditional DHV-1, the new type from Taiwan and the new type from Korea. These three types are also called duck Hepatitis A virus type 1 (DHV-1), type 2 (DHV-2) and type 3 (DHV-3), there is no cross protection or weak cross protection between each type. Duck Astro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/576G01N33/531
CPCG01N33/531G01N33/5768
Inventor 马波戚海惠刘悦张琪陈浩田常蕊张雪莲张文龙高明春王君伟
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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