Card for rapidly and quantitatively detecting pseudorabies virus antibody, and use method thereof

A pseudorabies virus, quantitative detection technology, applied in the field of detection cards, can solve the problems of low sensitivity and narrow detection range, and achieve the effects of high sensitivity, improved sensitivity and stable performance

Inactive Publication Date: 2018-03-30
杭州微瑞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] A large number of enzyme-labeled kits for detecting pseudorabies virus antibodies are used in the market. ELISA kits require a microplate reader, incubation reaction conditions, washing conditions, operating environment and professional technicians. In addition, detection samples also require complex pre-treatment, such as Collect blood, separate serum, etc.; while rapid colloidal gold detection is convenient and does not require professional technicians and working environment, but its detection sensitivity is low, and it can only be qualitative, and the detection range is narrow

Method used

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  • Card for rapidly and quantitatively detecting pseudorabies virus antibody, and use method thereof
  • Card for rapidly and quantitatively detecting pseudorabies virus antibody, and use method thereof
  • Card for rapidly and quantitatively detecting pseudorabies virus antibody, and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, eukaryotic expression of pseudorabies virus protein gB protein

[0039] Step 1. Primer design and PCR amplification of the target gene

[0040] Design specific upstream primer SP1: 5'-TCGAATTCATGAAAAAACTATTTGTG-3'; downstream primer SP2: 5'-TTGCGGCCGCTTACATCACATGGCGT-3' according to the nucleotide sequence of the porcine pseudorabies gB gene published in the GenBank database. location. Pichia P1: 5'-GACTGGTTCCAATTGACAAGC-3'; P2: 5'-GCAAATGGCATTCTGACATCC-3'. The synthetic gB genome clone was used as a template, primers SP1 and SP2 were used for PCR amplification reaction, and the final product was checked by 0.8% agarose gel electrophoresis.

[0041] Step 2. Construction and identification of cloning vector TS

[0042] After the target fragment was recovered by the gel recovery kit, it was ligated with the pBS-T vector at 16°C and transformed into Escherichia coli competent TOP10. After blue and white spot plating screening, PCR amplification, double enz...

Embodiment 2

[0054] Example 2. Antigens prepared by using specific synthetic peptides for pseudorabies virus proteins gB and gI (or gE) proteins

[0055] A method for preparing the above-mentioned pseudorabies virus structural protein gB protein, comprising the following steps:

[0056] Step 1. Sequence analysis of pseudorabies virus structural protein gB protein: use bioinformatics MHC class I molecular prediction: verify through relevant websites or use computer software to analyze the sequence of PRV-gB protein to understand its hydrophilicity, hydrophobicity, structure Domain accessibility, sequence variability, α-helix, β-turn, antigenicity and other parameters, and then comprehensive analysis and homology modeling methods to predict its tertiary structure, from which the antigenic reactive epitope and amino acid residues are predicted, The comprehensive analysis design is carried out according to the degree of difficulty of the peptide synthesizer. Each polypeptide chain contains at...

Embodiment 3

[0059] Embodiment three, the detection card that prepares pseudorabies virus antibody

[0060] Prepare the standard curve for the detection of pseudorabies virus gB antibody: prepare 6 copies of calibration solution containing pseudorabies virus antibody (including pseudorabies virus antibody standard), the concentrations are 0, 1 / 1024, 1 / 256, 1 / 64, 1 / 16. 1 / 4 (double dilution of pseudorabies virus antibody standard). Add the above-mentioned calibration solutions of different concentrations into the sample holes of the assembled test card, and after 15 minutes of chromatography, the test is carried out by a tomographic scanner, and the test results obtained 6 times are processed by the client, and the client calculates The fluorescence signal intensity values ​​of the detection line and quality control line corresponding to the standard product, and perform linear regression based on this data to make a standard curve of pseudorabies virus antibody. The standard curve calculat...

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Abstract

The invention discloses a card for rapidly and quantitatively detecting a pseudorabies virus antibody, and a use method thereof. The card comprises a detection card shell and a test strip assembled inthe detection card shell; the test strip comprises a plastic bottom plate with pressure-sensitive adhesive; a sample pad, a marker pad, a nitrocellulose film and water-absorbing paper are adhered tothe bottom plate sequentially; the marker pad consists of a carrier base layer and a marker; the marker is one layer of film formed by spray-coating the carrier base layer with a lanthanide fluorescence detection microsphere and a lanthanide fluorescence quality control microsphere; the nitrocellulose film is coated with a pseudorabies virus recombinant antigen serving as a detection line and coated with a rabbit anti-chicken IgY antibody serving as a quality control line; and the marker is a fluorescence detection microsphere marked with pseudorabies virus structure protein gB and gI (or gE)recombinant antigens and a fluorescence quality control microsphere marked with a chicken IgY antibody. The card can detect the pseudorabies antibody on site rapidly and quantitatively, and has higherpractical value and popularization value.

Description

technical field [0001] The invention relates to a detection card and its use, in particular to a rapid quantitative detection card for pseudorabies virus antibody in pig serum and its use method. Background technique [0002] Pseudorabies (PR) is a highly contagious disease caused by pseudorabies virus (PRV). PRV is a double-stranded DNA virus belonging to the herpesviridae herpesvirus subfamily A, herpesvirus type I. The complete virion is round, with a diameter of 150-180 nm, and the diameter of the nucleocapsid is 105-110 nm. There is a capsule, and on the surface of the capsule, there are fibrous processes about 8-10 nm in radial arrangement. [0003] The viral genome consists of a long unique region (Unique long region, UL), a short unique region (Unique short region, US), and a terminal repeat sequence (Wetminal repeat, TR) and an internal repeat sequence (Internal repeat, IR) located on both sides of US. According to the sequence determination of the PRV genome, it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983
Inventor 吴俊清章健吴冠英王泽洲
Owner 杭州微瑞科技有限公司
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