Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody

A porcine epidemic diarrhea and kit technology, which can be applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of large differences and the risk of whole virus antigen excretion, achieve good specificity, be easy to popularize and use in a large range, and simple to operate. Effect

Active Publication Date: 2014-03-26
GUANGXI UNIV
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Problems solved by technology

There is a potential risk of dispersal in the preparation of whole virus antigens, and most of the whole viruses are prepared from attenuated CV777 strains. Most studies have shown tha

Method used

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  • Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody
  • Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody
  • Indirect ELISA (enzyme linked immunosorbent assay) kit for detecting porcine epidemic diarrhea virus antibody

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Embodiment Construction

[0019] 1. Amplification of Nh sequence and construction of prokaryotic expression vector

[0020] Referring to the N gene primer designed by Yang Min (Master's thesis, Gansu Agricultural University, 2006) to amplify the porcine epidemic diarrhea nucleocapsid protein gene, the upstream primer for amplifying the N group is: 5'-CCGAGTGCGGTTCTCACAGAT-3'( Sequence listing SEQ.ID.No.4), the downstream primer is: 5'-CATAGCCAGGATAAGCCGGTC-3' (sequence listing SEQ.ID.No.5); PCR amplification of N Gene( figure 1 , Sequence Listing SEQ.ID.No.3). The PCR reaction system is 2.5 μL of 10×PCR Buffer, 0.25 μL of dNTPs (10 mM), 0.25 μL of Ex-Taq polymerase (5U / μL), 0.5 μL of upstream and downstream primers N1 / N2 (25pmol / μL), and sterile ddH2O Add to 25 μL. PCR reaction program: 95°C for 5min; 94°C for 1min, 48°C for 1min, 72°C for 1min30s, a total of 35 cycles; 72°C for 10min; 4°C for storage. The target gene was recovered and cloned into pMD-18T to obtain the cloning plasmid pMD-N. Then ...

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Abstract

The invention discloses an indirect ELISA (enzyme linked immunosorbent assay) kit for detecting a porcine epidemic diarrhea virus antibody. The indirect ELISA kit comprises a coated ELISA plate, negative control serum, positive control serum, ELISA secondary antibody, a concentrated cleaning solution, a sample diluent, a developing liquid and a stop buffer, wherein the coated ELISA plate utilizes recombinant protein Nh as a coating antigen. Research and practice show that the indirect ELISA kit has the characteristics of high specificity and sensitiveness, simplicity in operation and the like, is easy to popularize and use in a large range, has a broad market prospect, and can be used for the fields of serological investigation, antibody monitoring and vaccine immunity effect evaluation and the like of epidemic diarrhea infection condition.

Description

technical field [0001] The invention belongs to the technical field of ELISA kits, in particular to an indirect ELISA kit for detecting porcine epidemic diarrhea virus antibodies. Background technique [0002] Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDV) of the Coronaviridae family, and is characterized by diarrhea, vomiting, dehydration and high lethality of suckling piglets. The disease mainly harms suckling piglets, and the infection rate of piglets aged 2-7 days is high, mainly manifested as diarrhea, dehydration, vomiting, and depression. Piglets within 10 days of age are infected and have diarrhea for 2 to 4 days, and die in large numbers, which brings huge economic losses to the pig industry. In the 1980s and 1990s, many countries in Europe, including the United Kingdom, the Netherlands, France, Belgium, Germany and Switzerland, had the prevalence of PED. In recent years, the prevalence of PEDV in Europe has decreased; in contr...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/17
Inventor 黄伟坚郭旋龙凤
Owner GUANGXI UNIV
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