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An ELISA kit for detecting porcine atypical fever virus 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 antibodies against atypical fever virus of swine, etc., and achieves easy observation, high accuracy and low cost. low cost effect

Active Publication Date: 2019-07-19
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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  • An ELISA kit for detecting porcine atypical fever virus antibody based on e2 protein
  • An ELISA kit for detecting porcine atypical fever virus antibody based on e2 protein
  • An ELISA kit for detecting porcine atypical fever virus antibody based on e2 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] An ELISA kit for detecting ASFV antibody, the kit includes a reaction plate coated with ASFV E2 protein, enzyme-labeled antibody, chromogenic solution, stop solution, positive serum and negative serum.

[0025] Wherein, the preparation method of the reaction plate coated with ASFV E2 protein is as follows:

[0026] 1. Obtaining the E2 protein of ASFV:

[0027] 1. Cloning the E2 gene of swine atypical fever virus:

[0028] The primers for amplifying the E2 gene were designed according to the gene sequence of the APPV GD1 strain (GenBank: KX950761). 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 Yingweijieji (Shanghai) Trading Co., Ltd.

[0031] Serum samples were subjected to total RNA extraction according to the instructions of th...

Embodiment 2

[0061] Specificity Test of ELISA Kit for Detecting Antibody of Swine Atypical Fever virus

[0062] Positive sera from known viruses were tested for cross-reactivity using the method described in Example 1 to determine the specificity of the method. The results showed that the standard positive serum of swine fever virus (CSFV), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and circovirus (PCV) could be detected by this method. During the test, the ratio (P / N value) of all the sera to be tested 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] Repeatability test of ELISA kit for detection of swine atypical fever virus antibodies

[0067] Utilize the method described in Example 1 to carry out the repeatability test, the results show that the same sample is used for the repeatability test within and between batches, and the coefficient of variation is within 5%, indicating that the method has 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 present invention relates to the field of veterinary immune detection, and more particularly, to an ELISA kit for detecting swine atypical fever virus antibodies based on E2 protein. Background technique [0002] Piglet congenital tremor occurs in newborn piglets, which is characterized by skeletal myoclonic contractions and tremors after birth, and severe symptoms may lead to death due to starvation. In 2015, Hause's research group confirmed that Atypical porcine pestivirus (APPV) is the causative agent of congenital tremor in piglets. Subsequently, Germany, the Netherlands, Austria and other countries have successively discovered the prevalence of APPV in pigs. The research group of Ning Zhangyong from South China Agricultural University first discovered the occurrence of APPV infection in pigs in my country, and isolated APPV GD1 and GD2 strains, but so far the prevalence of APPV in pigs in my country is still unclear. At present, there is no ...

Claims

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Application Information

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Patent Type & Authority Patents(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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