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ELISA differential diagnosis kit, method and application of prrsv gene marker vaccine strain

A gene marker and differential diagnosis technology, applied in the fields of biochemical equipment and methods, biological testing, material inspection products, etc., can solve the problems of simultaneous existence of vaccines, indistinguishability, loss of aquaculture, and achieve strong specificity and good repeatability. , the effect of high sensitivity

Active Publication Date: 2016-09-14
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disease was first discovered in the United States in 1987, and PRRSV was isolated in my country for the first time in 1996. However, in 2006, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) brought huge losses to my country's breeding industry. Vaccine immunity It is the main means to prevent and control PRRSV at present. At present, the commercialized vaccines used to prevent PRRSV in China mainly include highly pathogenic PRRS inactivated vaccine, classic CH-1R attenuated vaccine, and three highly pathogenic PRRS attenuated vaccines. Vaccines and some foreign commercial attenuated vaccines, etc., the above-mentioned many types of PRRS vaccines are widely used in China. Although the use of vaccines can control the prevalence of PRRS, it also increases the difficulty of differential diagnosis of pig PRRS pathogenic infection
Therefore, it is necessary to develop new marker vaccines to solve the chaotic situation where many vaccines coexist and the highly pathogenic PRRSV and classic PRRSV coexist and are difficult to distinguish

Method used

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  • ELISA differential diagnosis kit, method and application of prrsv gene marker vaccine strain
  • ELISA differential diagnosis kit, method and application of prrsv gene marker vaccine strain
  • ELISA differential diagnosis kit, method and application of prrsv gene marker vaccine strain

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Experimental program
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Effect test

Embodiment 1

[0035] The preparation of embodiment 1 standard serum and the acquisition of clinical serum

[0036] 1. Preparation of recombinant NDV NP49 antigen protein

[0037] (1) Construction of recombinant expression NDV NP49 antigen prokaryotic expression vector

[0038] Design specific primers according to the published NDV La Sota strain (GenBank, NO.AF077761) NP protein gene sequence, upstream primer (NP49-UP): 5'-TTAGAATTCGGGGATGGGGAGACCCAAT-3' (SEQ ID NO: 2), its 5' An EcoR I restriction site is designed at the end; the downstream primer (NP49-DP): 5'-ATACTCG4GATACCCCCAGTCGGTGTCGT-3' (SEQ ID NO: 3), and an Xho I restriction site is designed at the 5' end. The expected amplified length is 165bp. The primers were synthesized by Invitrogen Company (Shanghai). The synthesized primers were diluted with sterilized double distilled water to a final concentration of 10 mmol / L, and stored at -20°C for use.

[0039] Total RNA of NDV La Sota strain was extracted according to the instruct...

Embodiment 2

[0055] Embodiment 2 NP49-ELISA experimental condition optimization

[0056] 1. Determination of the optimal coating concentration and serum dilution of the antigen

[0057] (1) Dilute the 10-fold gradient with 0.05M carbonate buffer (pH9.6), coat the NP49 polypeptide antigen (10 μg / mL to 0.125 μg / mL) on a 96-well plate, 100 μL per well;

[0058] (2) The coating condition is overnight at 4°C, and the sealing is carried out with 5% skimmed milk at 37°C for 2 hours.

[0059] (3) The positive serum and negative serum of PRRSV rHN4-Δ25+NP49 strain were serially diluted at 1:20, 1:40, 1:80, and 1:160, respectively, with 100 μL of dilution per well, and incubated at 37°C for 1 hour for ELISA method. array test.

[0060] (4) Horseradish peroxidase (HRP)-labeled goat anti-pig IgG antibody was diluted 1:10000 times, 100 μL per well, and incubated at 37°C for 1 hour.

[0061] (5) Add 50 μL of TMD chromogenic substrate, and develop color for 10 min at room temperature in the dark.

[...

Embodiment 3

[0092] Embodiment 3 specificity experiment

[0093] The established indirect NP49-ELISA method was used to detect clinically positive sera of CSFV, PRV, PPV, PCV-2, PEDV, and PRRSHuN4-F112. At the same time, PRRSV rHN4-Δ25+NP49 strain positive sera and negative sera were set up as controls to determine the NP49 polypeptide Whether the antigen cross-reacts with sera positive for other common swine diseases.

[0094] Results: OD was determined by detecting CSFV, PRV, PPV, PCV-2, PEDV, PRRS HuN4-F112 positive serum 450 The nm values ​​were 0.049, 0.075, 0.099, 0.044, 0.061, and 0.068, confirming that the NP49 polypeptide antigen had no cross-reaction with other common porcine disease-positive sera.

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Abstract

The invention discloses a kit for porcine reproductive and respiratory syndrome virus (PRRSV) gene-labeled vaccine strain ELISA differential diagnosis. The kit comprises NP49 polypeptide antigen and an amino acid sequence of the NP49 polypeptide antigen is shown in the formula of SEQ ID NO: 1. The kit for PRRSV gene-labeled vaccine strain ELISA differential diagnosis utilizes NP49 polypeptide as a coating antigen, has the characteristics of strong specificity, high sensitivity and good repeatability, can fast and accurately distinguish traditional PRRS vaccine- and labeled vaccine-immunized pig serum antibodies, can fast and accurately distinguish and diagnose the PRRSV gene-labeled vaccine strain and a natural infection strain, and has an important meaning for wide clinical application of the PRRSV gene-labeled vaccine strain.

Description

technical field [0001] The invention relates to the technical field of porcine reproductive and respiratory syndrome virus (PRRSV) detection, in particular to an ELISA differential diagnosis kit, method and application of a PRRSV gene-marked vaccine strain. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus, is a worldwide infectious disease that seriously harms the pig industry. The disease was first discovered in the United States in 1987, and PRRSV was isolated in my country for the first time in 1996. However, in 2006, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) brought huge losses to my country's breeding industry. Vaccine immunity It is the main means to prevent and control PRRSV at present. At present, the commercialized vaccines used to prevent PRRSV in China mainly include highly pathogenic PRRS inactivated vaccine, cl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01
CPCG01N33/56983G01N33/68
Inventor 童光志周艳君孙晶姜一峰童武
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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