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Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof

A duck hepatitis virus, enzyme-linked immunosorbent assay technology, applied in the field of poultry immunology, can solve the problems of low sensitivity, limited clinical application, long time required for detection, etc., and achieves good safety, great economy, and easy to popularize on a large scale. applied effect

Inactive Publication Date: 2010-09-22
HENAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there are deficiencies when these methods are used to detect the pathogen in dead duck tissues infected with DHV-I, such as the long time required for detection, low sensitivity, etc., the clinical application is limited to a certain extent

Method used

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  • Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof
  • Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof
  • Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof

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Embodiment Construction

[0063] 1. Primer design

[0064] According to the VP1 gene sequence of DHV-1 published by GenBank (sequence number: EF653378), use DNAStar software to analyze, design a pair of specific primers, and add restriction enzyme sites at both ends of the primers EcoR I and Xba I and protective bases (synthesized by Takara Company), the underlined part is the restriction site.

[0065] Upstream primer P1: 5′-CCA GAATTC GGTGATTCTAACCAG-3′ EcoR I

[0066] Downstream primer P2: 5′-GTT TCTAGA TTCAATTTCCAG-3′ Xba I

[0067] 2. PCR amplification of VP1 gene

[0068] PCR reaction conditions: preheating at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 55°C for 30 sec, extension at 72°C for 50 sec, 30 cycles, and extension for 10 minutes after the last cycle. After the PCR product was identified by electrophoresis on 1% agarose gel containing ethidium bromide (EB), the target band was excised, and then recovered according to the instructions of the gel r...

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Abstract

The invention relates to an enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody and relates to a test method and application of the kit. The kit comprises an enzyme label plate coated by the recombinant VP1 (virus protein) protein, a rabbit anti-duck IgY antibody marked by horseradish peroxidase, a TMB substrate colour reagent, a positive serum, a negative serum and a kit specification. In the invention, by adopting the polymerase chain reaction, the VP1 genes are amplified from the DHV-1genome and the VP1 gene-containing recombinant expression plasmid pET32a-VP1 is constructed; the plasmid is transferred to host cells BL21 (DE3), and the in-vitro expression VP1 protein is purified by a nickel column and then used as the antigen; the enzyme-linked immunosorbent assay kit is established; the positive serum is the standard positive serum of duck hepatitis virus type-I and the negative control is the standard negative serum of duck. The test kit has the advantages of strong specificity, high sensitivity, simple operation, easy large-scale popularization and application, very important application value in diagnosis of duck hepatitis virus type-I, survey of epidemiology and immunization survey and the like.

Description

technical field [0001] The invention relates to a type I duck hepatitis virus (Duckhepatitisvirus type-1, DHV-1) serum antibody ELISA detection kit, and also relates to a detection method and application of the kit, belonging to the technical field of poultry immunology . Background technique [0002] Duck viral hepatitis is a highly fatal acute infectious disease of young ducklings. The disease is characterized by acute onset, rapid spread, and high mortality. The clinical manifestations are opisthotonus, and the pathological changes are hepatitis and hemorrhage. , this disease often causes serious economic losses to duck farms. The disease was first discovered in the United States, and the virus was isolated from chicken embryos. Later, the disease was found in many duck-raising countries such as the United Kingdom, Canada, and Germany. Most provinces and regions of my country also have the occurrence of this disease and it is on the rise. duck hepatitis virus ( Duck h...

Claims

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Application Information

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IPC IPC(8): G01N33/576G01N33/535C12N15/51C12N15/70C07K14/085
Inventor 王臣赵战勤廖成水牛明福张春杰程相朝吴庭才李银聚
Owner HENAN UNIV OF SCI & TECH
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