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Enzyme linked immunosorbent assay (ELISA) kit for detecting atypical porcine pestivirus (APPV) antibody based on E2 protein

An atypical fever virus and kit technology, which is applied in the field of ELISA kits, can solve the problems of unclear APPV prevalence in pigs, lack of detection of atypical fever virus antibodies, etc., and achieve easy observation, high accuracy, and specificity. strong effect

Active Publication Date: 2018-02-06
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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  • Enzyme linked immunosorbent assay (ELISA) kit for detecting atypical porcine pestivirus (APPV) antibody based on E2 protein
  • Enzyme linked immunosorbent assay (ELISA) kit for detecting atypical porcine pestivirus (APPV) antibody based on E2 protein
  • Enzyme linked immunosorbent assay (ELISA) kit for detecting atypical porcine pestivirus (APPV) antibody based on E2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] An ELISA kit for detecting porcine atypical fever virus antibody, the kit comprises a reaction plate coated with porcine atypical fever virus E2 protein, an enzyme-labeled antibody, a color developing solution, a stop solution, positive serum and negative serum.

[0025] Wherein, the preparation method of the reaction plate coated with porcine atypical fever virus E2 protein is as follows:

[0026] 1. Acquisition of porcine SARS virus E2 protein:

[0027] 1. Cloning of porcine SARS virus E2 gene:

[0028] According to the gene sequence of APPV GD1 strain (GenBank: KX950761), primers were designed to amplify the E2 gene. The specific primers are as follows:

[0029] F1: 5′-GCCGTA GAATTC ATGTCATGCCACAGAAGACAAG-3' (SEQ ID NO: 3), underlined EcoR I restriction site;

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

[0031] Serum samples...

Embodiment 2

[0061] Specific Test of ELISA Kit for Detecting Porcine SARS Antibody

[0062] 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.

[0063] Table 1 Specificity test

[0064]

Embodiment 3

[0066] Reproducibility Test of ELISA Kit for Detecting Porcine SARS Antibody

[0067] 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).

[0068] Table 2 Repeatability test

[0069]

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Abstract

The invention discloses an enzyme linked immunosorbent assay (ELISA) kit for detecting an atypical porcine pestivirus (APPV) antibody based on E2 protein. The ELISA kit comprises a reaction plate enveloped by the E2 protein of the APPV shown in SEQ ID No. 1, an enzyme-labeled antibody, a color development solution, a stop solution, positive serum and negative serum. The invention creates the ELISAkit for detecting the APPV antibody for the first time and can rapidly, specifically and sensitively detect the APPV antibody in serum; the serum antibody detection sensitivity of the ELISA kit is 1to 1000. The ELISA kit provided by the invention is simple in use method, low in cost, easy in observation of reaction results and good in specificity and suitable for monitoring of APPV infection, epidemiological survey and detection of veterinary clinical samples, thus being suitable for wide-range popularization and application.

Description

technical field [0001] The invention relates to the field of veterinary immunoassay, more specifically, to an ELISA kit for detecting porcine atypical fever virus antibody based on E2 protein. 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, Hause's 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 宁章勇许古明刘健新郭世宁张凯照葛士坤
Owner SOUTH CHINA AGRI UNIV
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