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ELISA kit for detecting porcine atypical pestivirus antibody based on Npro protein

The technology of atypical plague virus and kit, applied in the field of ELISA kit, can solve the problems of lack of detection of porcine SARS virus antibody and unclear situation of APPV prevalence in swine herds, and achieve easy reaction results, high accuracy, good reproducibility

Active Publication Date: 2018-02-09
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ning Zhangyong's research group at South China Agricultural University discovered the occurrence of APPV infection in pigs in my country for the first time, and isolated APPV GD1 and GD2 strains, but the prevalence of APPV in pigs in my country is still unclear so far
At present, there is no vaccine available for porcine SARS virus infection, so there is currently a lack of relevant kits for detecting porcine SARS virus antibodies

Method used

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  • ELISA kit for detecting porcine atypical pestivirus antibody based on Npro protein
  • ELISA kit for detecting porcine atypical pestivirus antibody based on Npro protein
  • ELISA kit for detecting porcine atypical pestivirus antibody based on Npro protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] An ELISA kit for detecting porcine atypical fever virus antibody, said kit comprising porcine atypical fever virus N pro Protein-coated reaction plate, enzyme-labeled antibody, chromogenic solution, stop solution, positive and negative sera.

[0025] Among them, porcine atypical fever virus N pro Protein-coated reaction plates are prepared as follows:

[0026] 1. Porcine SARS virus N pro Protein acquisition:

[0027] 1. Cloning porcine atypical fever virus N pro Gene:

[0028] Design and amplify N according to the gene sequence of APPV GD1 strain (GenBank: KX950761) pro Gene primers, the specific primers are as follows:

[0029] F1: 5′-GCGGCG GAATTC ATGAAGAAGCAGATTACAT -3′ (SEQ ID NO: 3), the underline indicates the EcoRI restriction site;

[0030] R1: 5′-GCGATG CTCGAG TTAACAGGTCTTCACATAAAGC-3' (SEQ ID NO: 4), underlined xho I restriction site. Primers were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd.

[0031] Serum samples were subjected to ...

Embodiment 2

[0060] Specific Test of ELISA Kit for Detecting Porcine SARS Antibody

[0061] The method described in Example 1 was used to test the cross-reactivity of known positive sera of various viruses to determine the specificity of the method. The results showed that the method was effective against standard positive sera of classical swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and circovirus (PCV). When tested, the ratio (P / N value) of all tested sera to APPV-negative sera was less than 2.1 (Table 1), indicating that the method has good specificity.

[0062] Table 1 Specificity test

[0063]

Embodiment 3

[0065] Reproducibility Test of ELISA Kit for Detecting Porcine SARS Antibody

[0066] Utilize the method described in embodiment 1 to carry out repeatability test, the result shows, utilize this experimental method to carry out repeatability test in batch and between batches to same sample, coefficient of variation is all within 5%, illustrates that this method has very high repeatability (Table 2).

[0067] Table 2 Repeatability test

[0068]

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Abstract

The invention discloses an ELISA kit for detecting a porcine atypical pestivirus antibody based on an Npro protein. The kit comprises a reaction plate coated with porcine atypical pestiviruses havingsequences shown in the formula of SEQ ID NO. 1, an enzyme-labeled antibody, a developing solution, a stop buffer, positive serum and negative serum. The invention creates the ELISA kit for detecting aporcine atypical pestivirus antibody. The kit can fast, specifically and sensitively detect the porcine atypical pestivirus antibody and has antibody detection sensitivity of 1: 1000. The kit is easyto use, realizes a low cost, reaction result observation easiness and good specificity, is suitable for the monitoring of the atypical pestivirus infection, epidemiological investigation and clinicalveterinary sample detection and can be widely used.

Description

technical field [0001] The present invention relates to the field of veterinary immunoassay, more specifically, to a N-based pro ELISA kit for protein detection of porcine SARS antibody. Background technique [0002] Piglet congenital tremor occurs in newborn piglets, characterized by the observation of skeletal myoclonic contraction and tremor after birth, and severe symptoms may lead to death due to starvation. In 2015, the Hause research team confirmed that Atypical porcine pestivirus (APPV) is the causative agent of congenital tremor in piglets. Subsequently, many countries such as Germany, the Netherlands, and Austria successively discovered the prevalence of APPV in pig herds. The Ning Zhangyong research group of South China Agricultural University discovered the occurrence of APPV infection in pigs in my country for the first time, and isolated APPV GD1 and GD2 strains, but the prevalence of APPV in pigs in my country is still unclear so far. Currently, there is no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/005
Inventor 宁章勇刘健新许古明郭世宁张凯照葛士坤
Owner SOUTH CHINA AGRI UNIV
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