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Kit for quickly detecting swine fever antibody and preparation method thereof

A technology of swine fever antibody and kit, which is applied in the field of swine fever antibody detection kit and its preparation, can solve the problems of complicated operation technology and poor repeatability of swine fever antibody, and achieve high sensitivity, broad market prospect and social Benefits, showing the effect of science

Active Publication Date: 2011-09-07
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current detection methods have poor reproducibility and cumbersome operation techniques for the detection of swine fever antibodies.

Method used

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  • Kit for quickly detecting swine fever antibody and preparation method thereof
  • Kit for quickly detecting swine fever antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 prepares classical swine fever virus specific antigen

[0035] According to the CSFV E2 gene sequence, a pair of primers are designed to amplify the main antigenic epitope region sequence of the E2 gene. Among them, the upstream primer P1 is: 5'-TAAAA GGATCC GGCCTGACCACCCACCTGGGAAG-3',

[0036] The downstream primer P2 sequence is: 5'-CCCCT CTCGAG TTAATAGCTATCACGCGGTTCATAATATTTG-3'.

[0037] Amplify the main epitope region sequence of the E2 gene of the CSFV Shimen strain (purchased from China Veterinary Microbiology Culture Collection and Management Center, virus numbering CVCC AV1411) by RT-PCR method,

[0038] Specific steps:

[0039] The reverse transcription reaction system is as follows: first mix 2 μL of downstream primer P2 (25 μmol / L) with total RNA of CSFV Shimen strain, bathe in water at 70°C for 10 minutes, then rapidly ice-bath for 5 minutes; then add dNTP mixture (concentrations are 10 mM, TaKaRa, Dalian, China) 4 μL, RNase inhibitor (40...

Embodiment 2

[0043] Embodiment 2 Preparation of kit of the present invention

[0044] Antibody detection kit for classical swine fever, including (1) enzyme-linked plate coated with specific antigen of classical swine fever virus, (2) rabbit anti-pig IgG (immunoglobulin) labeled with horseradish peroxidase (HRP), (3 ) sample diluent, (4) TMB chromogen, (5) stop solution, (6) 20 times concentrated washing solution, (7) negative control, (8) positive control.

[0045] CB coating solution: pH9.6, the specific composition and concentration are: anhydrous sodium carbonate (Na 2 CO 3 ) 1.59g / l, sodium bicarbonate (NaHCO 3 ) 2.93g g / l.

[0046] 20 times concentrated lotion: disodium hydrogen phosphate 12H 2 O 21g / l, sodium dihydrogen phosphate 2H 2 O 2.80g / l, sodium chloride 170g / l, Tween 20ml / l.

[0047] 10mM PBS pH7.4 Formula: Disodium Hydrogen Phosphate·12H 2 O 2.2g / l, sodium dihydrogen phosphate · 2H 2 O 0.2g / l, sodium chloride 8.5g / l

[0048] TMB Chromogen: The concentration of TMB ...

Embodiment 3

[0065] Implementation example 3 The method that kit of the present invention detects swine fever antibody

[0066] Test sample preparation:

[0067] ① Blood collection, static sedimentation or 3000 rpm centrifugation for 5 minutes, the supernatant is the serum.

[0068] ② Collect blood, add anticoagulant, centrifuge at 3000 rpm for 5 minutes, and the supernatant is plasma.

[0069] The sample should be light yellow, yellow serum or plasma without chyle, hemolysis and foreign matter. Various commonly used anticoagulants had no effect on the test results. If not tested immediately, it can be stored in a refrigerator at 2-8°C for 1-2 days. Long-term storage needs to be frozen at -18°C to -25°C, equilibrate to room temperature and mix well before testing.

[0070] The specific operation steps are as follows:

[0071] 1. Equilibration: Take the kit out of the refrigerated environment and equilibrate at room temperature for 30 minutes before use.

[0072] 2. Prepare lotion: di...

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Abstract

The invention relates to a kit for quickly detecting a swine fever antibody. In the method, a pair of specific primers is designed, a relatively conservative gene sequence is cloned, an antigen aiming at a swine fever E2 protein is expressed through a pronucleus expression technology, and, on the basis, the kit containing an enzyme linked plate coated with a high-purity and high activity swine fever virus specificity antigen, an enzyme conjugate of rabbit-anti-swine monoclonal antibody containing an HRP (Horseradish Peroxidase) marker, a TMB (Tetramethylbenzidine) color developing liquid and the like is prepared. The kit can quickly detect the swine fever antibody in blood serum or blood plasma and has strong specificity and high sensitivity.

Description

technical field [0001] The invention relates to a swine fever antibody detection kit and a preparation method thereof, which are used for detecting swine fever antibodies in pig serum or plasma samples, and belong to the field of enzyme-labeled immunoassay. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease caused by swine fever virus (CSFV). It was first discovered in Ohio, USA in 1833. It was once widely prevalent in pig-raising countries in the world. It causes serious harm and is one of the notifiable diseases determined by the World Organization for Animal Health (OIE). For many years, CSF has also endangered the development of my country's pig industry. It is one of the four major animal diseases in my country and is legally classified as a first-class infectious disease by the Ministry of Agriculture of my country. CSF is an acute and severe infectious disease with a mortality rate as high as 80% to 90%. Vaccination is the only e...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/531
Inventor 薛峰高海岗张小荣蒋原吴艳涛张睿刘秀梵赵红霞
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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