ELISA kit for detecting porcine epidemic diarrhea virus pandemic strain antibody

A technology for porcine epidemic diarrhea and epidemic strains, which is applied in biological testing, measuring devices, material inspection products, etc., and can solve problems such as specificity to be verified

Inactive Publication Date: 2014-11-19
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many scholars have researched and explored the PEDV ELISA detection method, the commercialized PEDV detection kits are all coated with PEDV whole virus antigen, and their specificity has yet to be verified during the detection process.

Method used

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  • ELISA kit for detecting porcine epidemic diarrhea virus pandemic strain antibody
  • ELISA kit for detecting porcine epidemic diarrhea virus pandemic strain antibody
  • ELISA kit for detecting porcine epidemic diarrhea virus pandemic strain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 The acquisition of coated antigen S recombinant protein

[0018] 1. Method

[0019] 1.1 Design and synthesis of 966 gene primers at the 5′ end of PEDV S gene

[0020] According to the gene sequence of PEDV CV777 (AF353511) strain published on GenBenk, a pair of primers were designed using Primer5.0 software to amplify the 5' end part of PEDV S gene. The primer sequences are as follows, synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0021] SP1: 5'-AACCATGGAAGATG TCACTAGGTGCC-3', SEQ ID NO: 3

[0022] SP2: 5'- ATACTGCTTTAGGAACAAATCTTGAGCTCCC-3', SEQ ID NO: 4

[0023] 1.2 Cloning and expression of PEDV S gene 966 gene protein

[0024] From the diarrhea of ​​suspected PEDV clinical pigs, PCR amplification was verified as PEDV infection. SP1 and SP2 were used as primers to amplify the 966bp gene at the 5' end of the PEDV S gene, cloned into the prokaryotic expression vector pET-28a, and verified by enzyme digestion After the positive clone was sent ...

Embodiment 2

[0054] Embodiment 2, preparation and use of kit

[0055] 1. The composition of the kit is shown in Table 3.

[0056] Table 3 Kit preparation composition

[0057] Composition (96tests×5 / box)

quantity

Enzyme-labeled reaction plate coated with S recombinant protein

5 pieces

serum diluent

100ml

10×washing liquid

200ml

negative control

100μl

positive control

100μl

Chromogenic Solution A and Chromogenic Solution B

30mL each

Horseradish peroxidase-labeled rabbit anti-pig IgG

60ml

stop solution

60mL

[0058] 2. How to use the kit

[0059] (1) Dilute serum with 96-well cell plate: add serum diluent to each well of 96-well cell plate, 196 μl / well. Add 4 μl of PEDV-positive serum to wells A1 and B1, and add 4 μl of PEDV-negative serum to wells C1 and D1. Add 4 μl of serum to be tested to the remaining wells. After mixing evenly, take 100 μl with a pipette and transfer in paral...

Embodiment 3

[0067] Embodiment 3, test kit repeatability detection result

[0068] 7 batches of antigen-coated ELISA kits from the same batch were repeatedly tested within the batch, and the test results showed that the coefficient of variation of the repeated tests within the batch was 1.16%-6.6%.

[0069] 7 batches of antigen-coated ELISA kits from different batches were repeatedly tested, and the test results showed that the coefficient of variation of repeated tests between batches was 1.7%-7.7%, all lower than 8%.

[0070] It is proved that the repeatability of the ELISA detection kit is good.

[0071] Table 4 7 batches of ELISA kits repeated test results within batches and between batches

[0072]

[0073]

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Abstract

The invention discloses an ELISA kit for detecting a porcine epidemic diarrhea virus pandemic strain antibody. The kit comprises an enzyme mark reaction plate coated with antigen, a washing liquid, a weak solution, a horseradish peroxidase labelled rabbit anti-pig IgG second antibody, a coloured solution and a stopping solution; wherein the coating antigen is S recombinant protein of porcine epidemic diarrhea virus pandemic strain. The kit is capable of rapidly detecting the porcine epidemic diarrhea virus pandemic strain antibody with safe usage.

Description

technical field [0001] The invention relates to a reagent kit for detecting antibodies to epidemic strains of porcine epidemic diarrhea virus by using an ELISA method, and belongs to the field of animal inspection and quarantine. Background technique [0002] Since the second half of 2010, a new round of outbreaks of porcine epidemic diarrhea has occurred in my country. It first occurred in Guangxi, and then became widespread in Hunan, North China, East China, Shandong, Hebei and other places. It became a nationwide pandemic and caused a large number of deaths of suckling piglets. According to statistics from the Epidemiology Center of the Ministry of Agriculture, the number of pig deaths caused by porcine epidemic diarrhea (PED) disease in 2011 has occupied the first place among all existing pig diseases in my country. [0003] Studies have shown that the S gene, M gene, and E gene of PEDV strains in different regions have significant differences, especially the S gene has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/6803G01N2333/183
Inventor 王一成袁秀芳徐丽华李军星
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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