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ELISA kit for distinctively detecting antibodies of classical swine fever (CSF) vaccine immunity and wild virus infection and preparation method thereof

A swine fever vaccine and kit technology, applied in measuring devices, instruments, scientific instruments, etc., can solve problems such as difficulty in ensuring accuracy, difficulty in collection, difficulty in large-scale promotion and application, etc.

Inactive Publication Date: 2010-12-01
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the CSFV virus nucleic acid differential detection technology also has great deficiencies: on the one hand, due to the low amount of CSFV virus in pig blood, the detection rate of blood virus nucleic acid is often low; although the tonsil virus content is high, it requires special equipment and collection. Difficult; on the other hand, RT-PCR and fluorescent PCR detection require special equipment, environment and personnel, the detection cost is high, and it is difficult to popularize and apply on a large scale; and the experimental results are also vulnerable to cross-contamination, nucleic acid extraction efficiency, RNA reverse transcription Affected by multiple factors such as efficiency, accuracy is difficult to guarantee

Method used

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  • ELISA kit for distinctively detecting antibodies of classical swine fever (CSF) vaccine immunity and wild virus infection and preparation method thereof
  • ELISA kit for distinctively detecting antibodies of classical swine fever (CSF) vaccine immunity and wild virus infection and preparation method thereof
  • ELISA kit for distinctively detecting antibodies of classical swine fever (CSF) vaccine immunity and wild virus infection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1 Prokaryotic expression and purification of NS3

[0088]1. Viral RNA extraction: extract the total RNA of hog fever rabbitization attenuated cell culture fluid with TRIzol Reagent kit (purchased from Invitrogen);

[0089] 2. Design and synthesis of primers: Design a pair of primers based on the NS3 gene sequence published in GenBank, and add Sac I and Xho I restriction sites at both ends of the primers respectively, to amplify the 2049 bp gene between 5142 and 7191 bp of the open reading frame fragment. The primers (sequence 1 and sequence 2) sequences are respectively:

[0090] P1: 5' GAG CTC GGG CCT GCC GTT TGC AA-3';

[0091] P2: 5' CTC GAG CTA GAC CAA CTA CCT GTT TTA GTG CTC TG-3';

[0092] The underlined part is the restriction site introduced, and the primers were synthesized by Invitrogen.

[0093] 3. Amplification and recovery of NS3 gene fragments

[0094] (1) RT-PCR amplification: use the extracted RNA as a template, and use the primers in ...

Embodiment 2

[0105] Example 2 Eukaryotic expression and purification of NS3

[0106] 1. Viral RNA extraction: extract the total RNA of hog fever rabbitization attenuated cell culture fluid with TRIzol Reagent kit (purchased from Invitrogen);

[0107] 2. Primer design and synthesis: Design a pair of primers based on the NS3 gene sequence published by GenBank, and add EcoR I and Xho I restriction sites at both ends of the primers, which can amplify the gene of 2049 bp between the open reading frame 5142 and 7191 bp fragment. The sequences of the primers (SEQ ID NO: 3 and SEQ ID NO: 4) are:

[0108] P3: 5'- GAA TTC GGG CCT GCC GTT TGC AAG-3';

[0109] P4: 5'- CTC GAG CTA GAC CAA CTA CCT GTT TTA GTG CTC-3';

[0110] The underlined part is the restriction site introduced, and the primers were synthesized by Invitrogen.

[0111] 3. Amplification and recovery of NS3 gene fragments

[0112] (1) RT-PCR amplification: use the extracted RNA as a template, and use the primers in step 2 for ...

Embodiment 3

[0126] Example 3 NS3 Antibody Detection Negative and Positive Control Serum Preparation and Reagent Preparation

[0127] 1. Preparation of negative and positive control serum for NS3 antibody detection

[0128] (1) Positive control serum for NS3 antibody detection of swine fever vaccine: Collect blood and separate serum every week after healthy non-immune pigs are immunized with swine fever rabbit attenuated vaccine, collect 10 times in total, combine and mix well as swine fever vaccine immunization NS3 antibody detection Positive control serum.

[0129] (2) Positive control serum for NS3 antibody detection of CSF wild virus infection: After the healthy non-immune pigs were inoculated with low-virulence and moderate-virulence CSFV, blood was collected every week to separate the serum, and the serum was collected 10 times in total, combined and mixed as CSF Positive control serum for wild virus infection NS3 antibody detection.

[0130] (3) Negative control serum for CSF vacc...

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Abstract

The invention relates to an ELISA kit for distinctively detecting antibodies of classical swine fever (CSF) vaccine immunity and wild virus infection and a preparation method thereof. An indirect ELISA kit or a blockage ELISA kit is formed by expressing and purifying classical swine fever virus (CSFV) non-structural protein NS3, coating a solid-phase carrier and assembling with other matched reagents. The kit has the characteristic of distinctively detecting different antibodies generated by the CSF vaccine immunity and the wild virus infection. The kit can distinctively diagnose the CSF vaccine immunity and the wild virus infection.

Description

technical field [0001] The invention relates to a differential detection method for swine fever antibodies, belonging to the technical field of veterinary biological products. Background technique [0002] Classical Swine Fever (CSF) is a highly contagious and fatal swine infectious disease caused by Classical Swine Fever Virus (CSFV), which occurs in many countries and regions in the world. Industrial production has brought serious economic losses. The World Organization for Animal Health (OIE) lists swine fever as a statutory notifiable disease, and my country classifies it as a first-class animal infectious disease and includes it in the major animal disease prevention and control plan. [0003] In the world, there are mainly 3 kinds of live vaccines with good effect on swine fever that can be used for the prevention and control of swine fever, namely the GPE strain in Japan, the Thiverval strain in France and the attenuated rabbit strain (C strain) in China, among which...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
Inventor 戴志红王在时关孚时蒋卉李翠赵耘秦玉明
Owner CHINA INST OF VETERINARY DRUG CONTROL
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