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Indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting porcine reproductive and respiratory syndrome virus

A technology for respiratory syndrome and immune reagents, applied in the field of biological vaccine preparation, can solve the problems of long diagnosis time, large workload, insufficient specificity and sensitivity, and achieve simple operation, short time required, and good consistency. Effect

Inactive Publication Date: 2013-09-11
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above technologies either have too much workload, or need to operate live viruses, or have insufficient specificity and sensitivity, or the diagnosis time is too long, which affects the control of the disease, or cannot be conveniently used for clinical testing.

Method used

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  • Indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting porcine reproductive and respiratory syndrome virus
  • Indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting porcine reproductive and respiratory syndrome virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation method of the kit:

[0041] 1. Preparation and coating of nucleoprotein expressed by porcine reproductive and respiratory syndrome virus prokaryotic expression system:

[0042]The recombinant nucleoprotein of part or all of the porcine reproductive and respiratory syndrome virus nucleoprotein gene was inoculated into the Escherichia coli expression system and cultured at 37°C for 16 hours. The culture was collected for recombinant protein purification and diluted to 100 μg / ml with coating solution when coating 100 μl / well was added to the enzyme-linked plate, and coated overnight at 4°C; washed 3 times with PBST, and 100 μl of blocking solution (5% skimmed milk powder and 0.01% thimerosal) was added to each well, and left at 37°C for 1 hour. Discard the washing solution, wash 3 times with PBST, vacuum seal with an aluminum foil bag, and store at 4°C.

[0043] 2. Preparation of standard serum:

[0044] Pigs were immunized twice with PRRSV virus purified in ...

Embodiment 2

[0063] Preparation method of the kit:

[0064] 1. Preparation and coating of nucleoprotein expressed by porcine reproductive and respiratory syndrome virus eukaryotic expression system:

[0065] Inoculate and transfect CHO-K1 cells with the recombinant nucleoprotein of part or all of the nucleoprotein gene of porcine reproductive and respiratory syndrome virus, culture at 37°C for 2 to 3 days, collect the cell culture, freeze and thaw twice, harvest the culture medium, pack Dilute to 100 μg / ml with coating solution, add 100 μl / well to the enzyme-linked class, coat at 4°C overnight; wash with PBST 3 times, add 100 μl blocking solution (5% degreased) to each well milk powder and 0.01% thimerosal), and placed at 37°C for 1 h. Discard the washing solution, wash 3 times with PBST, vacuum seal with an aluminum foil bag, and store at 4°C.

[0066] 2. Preparation of standard serum:

[0067] Pigs were immunized twice with PRRSV virus purified in the laboratory. Before immunization a...

Embodiment 3

[0086] 1. Specificity test:

[0087] Use the established competitive ELISA (NA N002-ELSA) method to detect 30 porcine foot-and-mouth disease positive sera, porcine parvovirus positive sera, porcine circovirus-2 and porcine vesicular disease virus sera, and simultaneously porcine reproductive and respiratory syndrome virus 30 standard positive sera and 30 negative quality control sera were used as positive and negative controls to analyze the specificity of the method. Positive porcine foot-and-mouth disease serum, porcine parvovirus positive serum, porcine circovirus-2 type, porcine vesicular disease virus serum and negative serum were all negative, only porcine reproductive and respiratory syndrome virus standard positive serum showed positive results, The results are shown in Table 5 below. Table 5 shows that the specificity test of the established PRRSV competition ELISA shows that it has good specificity, which fundamentally ensures the accuracy and reliability of the dete...

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Abstract

The invention discloses an indirectly competitive ELISA (Enzyme Linked Immunosorbent Assay) immune kit for detecting a porcine reproductive and respiratory syndrome virus. The kit comprises an enzyme marker for resisting a monoclonal antibody of the porcine reproductive and respiratory syndrome virus. The kit has the beneficial effects that the antibody valence of the porcine reproductive and respiratory syndrome virus is detected accurately and quantitatively; the kit is operated simply and short in required operation time. Thus, the kit can be used for detecting a large number of samples and surveying epidemiology, and is well consistent with a testing method recommended by WTO (World Health Organization) and OIE (Office International Des Epizooties).

Description

technical field [0001] The invention relates to the technical field of biological vaccine preparation, in particular to an indirect competition ELISA immune kit for detecting porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) causes enormous economic losses to the swine industry worldwide. The disease first appeared in Germany and the United States almost simultaneously in the 1980s, and since then, PRRS has broken out and spread worldwide. In the spring of 2006, the outbreak of "unknown high fever" in central China, that is, highly pathogenic porcine reproductive and respiratory syndrome (PHFD), caused widespread concern in the society with a mortality rate as high as 20% to 100%. [0003] Porcine reproductive and respiratory syndrome virus (PRRSV) is the main cause of the disease. It belongs to the Arteriviridae family, Arterivirus genus. Pigs of all ages are susceptible. Infected pregn...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 刘永生张杰丁耀忠陈豪泰周建华马丽娜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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