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

A gene marker and differential diagnosis technology, applied in biological testing, material testing products, measuring devices, etc., can solve the problem of inability to distinguish between wild virus and vaccine virus infection, and achieve the effect of simple structure, small molecular weight and high sensitivity

Active Publication Date: 2016-08-03
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccines used in China mainly include attenuated vaccines and inactivated vaccines. Among them, attenuated vaccines are widely used in clinical practice to control the prevalence of PRRS due to their characteristics of rapid antibody production, long duration and strong protection. However, traditional attenuated vaccines Unable to distinguish wild and vaccine virus infection during antibody surveillance

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Determination of Example 1 Antigen Optimal Coating Concentration and Optimum Serum Dilution

[0027] 1. Sera and Reagents

[0028] PRRS negative serum, PRRSV HuN4-F112 positive serum, PRRSVrHN4-Δ25+NP49 strain positive serum, porcine pseudorabies (PRV) positive serum, classical swine fever (CSFV) positive serum and porcine epidemic diarrhea (PEDV) positive serum are all in this experiment Room preservation. Porcine parvovirus (PPV) positive serum and porcine circovirus type 2 (PCV-2) positive serum were provided by the Porcine Disease Laboratory of Harbin Veterinary Research Institute;

[0029] Horseradish peroxidase (HRP)-labeled goat anti-pig IgG was purchased from Sigma Company; skim milk was purchased from BD Company; TMB chromogenic solution, skim milk, and bovine serum albumin (BSA) were purchased from AMRESCO Company. PRRSV antibody detection kit HerdCheckPRRSX3 was purchased from IDEXX company.

[0030] 2. Determination of the optimal coating concentration an...

Embodiment 2

[0036] The determination of embodiment 2 optimal reaction conditions

[0037] 1. Method

[0038] Optimize the reaction conditions from the aspects of coating time, blocking solution selection, blocking time, serum dilution, enzyme-labeled secondary antibody dilution, substrate reaction time, etc., other conditions remain unchanged, use PRRSHuN4-F112 positive serum and negative serum Carry out indirect 25aa-ELISA assay, read OD with enzyme-linked detector 450 nm value, compare the OD of negative and positive serum in each group 450 nm value and P / N value to determine the best reaction conditions.

[0039] 2. Results

[0040] 2.1 Determination of blocking solution

[0041] Choose 5% skimmed milk, 1% BSA, 2% BSA, 10% fetal bovine serum as the blocking solution. Compare the OD of negative and positive serum in each group 450 nm value and P / N value (Table 2), when 5% skim milk is used as blocking solution, negative serum OD 450 The nm value is the smallest, and the P / N value...

Embodiment 3

[0069] The determination of embodiment 3 critical value

[0070] Collect serum from clinical animal experiments, and measure OD according to the 25aa-ELISA method with optimized conditions 450 nm value, calculate the S / P value of the serum sample according to the following formula:

[0071] S / P=(OD of serum sample to be tested-OD of negative control) / (OD of positive control-OD of negative control).

[0072] Samples with S / P value ≥ critical value are judged as positive, and those with S / P value < critical value are judged as negative. The critical value is obtained by ROC statistical analysis method when the sum of sensitivity and specificity values ​​is the largest Corresponding S / P value.

[0073] The ROC curve is a comprehensive index reflecting the continuous variables of sensitivity and specificity. The ROC curve was drawn by SPSS software to obtain the sensitivity and specificity of the 25AA-ELISA detection method. After data processing and analysis, the negative and ...

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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 25aa polypeptide antigen and an amino acid sequence of the 25aa polypeptide is shown in the formula of SEQ ID NO: 1. The kit for PRRSV gene-labeled vaccine strain ELISA differential diagnosis utilizes PRRSV gene-labeled vaccine strain (rHN4-delta25+NP49 strain) specifically labeled zone-lost 25aa 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 an infectious disease characterized by reproductive disorders and respiratory diseases in sows of all ages was discovered in the United States and Canada, and spread rapidly to all parts of the world; in 1996, my country isolated PRRSV for the first time, and in 2006 The large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) in 2010 has brought huge losses to the breeding industry in my country. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/543
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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