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Rapid detection test strip for BVDV (bovine viral diarrhea virus) antibody and preparation method thereof

A bovine viral diarrhea and antibody detection technology, which is applied in the field of animal virology and zoonosis, can solve the problems of BVDV clinical detection difficulties, persistent infection, etc., and achieve easy storage and transportation, low detection cost, good specificity and The effect of sensitivity

Inactive Publication Date: 2013-07-10
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, these methods are not suitable for on-site testing and clinical testing in primary veterinary diagnostic departments
In addition, because BVDV can cause persistent infection, it brings great difficulties to the clinical detection of BVDV

Method used

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  • Rapid detection test strip for BVDV (bovine viral diarrhea virus) antibody and preparation method thereof
  • Rapid detection test strip for BVDV (bovine viral diarrhea virus) antibody and preparation method thereof
  • Rapid detection test strip for BVDV (bovine viral diarrhea virus) antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Expression of truncated BVDVNS3 protein

[0023] (1) BVDV NS protein antigen epitope analysis and target protein screening: By searching the BVDV NS protein sequence (NP_776266) and using online software to analyze its main antigen epitope, the selected antigen epitope is located at the 165th to 425th position in the NS3 protein Amino acids are used as target protein fragments, and its sequence is SEQ ID No.1 in the sequence listing. The corresponding nucleotide sequence is the 4286th to 5068th nucleotides in the BVDV genome sequence (NC_001461.1) published by GeneBank.

[0024] (2) Design of NS primers: According to the 4286-5068 nucleotide sequence of the gene fragment of the BVDV genome sequence (No. NC001461.1) published by GeneBank, specific primers for amplifying the BVDV NS3 gene were designed. ' end to add LIC cohesive ends. The primer sequences are as follows:

[0025] Upstream primer (P1): 5′-CAGGGACCCGGT-CTGCCTACCTATGAATTGG-3′

[0026] Downstre...

Embodiment 2

[0040] Embodiment 2: the preparation of colloidal gold label test strip

[0041] (1) Preparation of colloidal gold and labeling SPG with colloidal gold: Colloidal gold solution was prepared by trisodium citrate reduction method. Add 1mL of 1% chloroauric acid solution to 99mL of three-distilled water, heat to boiling, quickly add 2.4mL of freshly prepared 1.05% (m / v) trisodium citrate solution, mix well, continue heating for about 5-10min, The color of the solution changes from blue to red. After cooling to room temperature, filter with a 0.22 μm filter membrane. First adjust the pH value of the colloidal gold solution to 6.5 with 1% sodium carbonate solution. Slowly add 20 μL of SPG solution with a concentration of 1 mg / mL to 50 mL of colloidal gold solution, stir at room temperature for 30 min, slowly add 10% ovalbumin (OVA) to a final concentration of 10 mg / mL, and continue stirring for 30 min. Centrifuge at 4,000r / min for 10min, transfer the supernatant to another centr...

Embodiment 3

[0047] Embodiment 3: specificity, sensitivity and coincidence rate test of test strip

[0048] (1) Specificity of test strips: Use test strips to detect standard positive serum samples of BVDV (type 1 and type 2), other related viruses including swine fever virus (CSFV), Akabane disease virus (AKV), border disease Virus (BDV), type O foot-and-mouth disease virus (FMDV O), bluetongue virus (BTV), Peste des petits ruminants virus (PPRV) positive serum samples and negative control samples. The result shows that the test strip is positive when detecting BVDV (type 1 and type 2) standard positive serum samples, and is negative when other virus positive serum and negative control samples are tested. It shows that the specificity of the test strip is good.

[0049] (2) Sensitivity of test strips: test 5 serially diluted BVDV positive serum samples (numbered 1 to 5) with test strips, and use imported ELISA kits as a control, the results show that 5 samples were detected by test strip...

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Abstract

The invention discloses a recombinant antigen for BVDV (bovine viral diarrhea virus) antibody detection and a rapid detection test strip thereof, mainly aiming at a truncated BVDV NS expression antigen for BVDV antibody detection and a rapid detection test strip for detecting BVDV antibodies. A BVDV NS protein amino acid sequence is analyzed by utilizing epitope analysis software; a fragment with the main epitope is taken; a truncated BVDV NS recombinant antigen is obtained according to the corresponding nucleotide sequence design primer through PCR (polymerase chain reaction) amplification, recombination expression carrier construction and prokaryotic expression, and can be used for BVDV antibody detection after being purified. The rapid detection test strip established based on the truncated BVDV NS antigen has the advantages of convenience in operation, rapid detection, no requirement of a special laboratory and equipment and the like, overcomes the limits of the existing detection method, and can be used for rapid detection and serum epidemiological investigation of the BVDV antibodies.

Description

technical field [0001] The invention relates to the fields of animal virology and animal infectious disease, in particular to a rapid detection test strip for detecting bovine viral diarrhea virus antibody and a preparation method thereof. Background technique [0002] Bovine viral diarrhea (BVD), also known as bovine viral diarrhea / mucosal disease (BVD-MD), is caused by bovine viral diarrhea virus (BVDV) It is a contagious disease mainly occurring in cattle. BVDV, classical swine fever virus (CSFV), sheep border disease virus (BDV) and some unclassified pestiviruses belong to the genus Pestivirus in the family Flaviviridae, and they are single-stranded positive-sense RNA viruses. Viruses in members of the Pestivirus genus were considered host-specific in the past, but subsequent studies have shown that BVDV and BDV can infect a wider range of hosts. They can infect not only cattle and sheep, but also a variety of artiodactyly, including pigs. class animals. [0003] At p...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 杨俊兴花群义曹琛福张彩虹吕建强卢体康孙洁陈兵阮周曦秦智锋刘建利胡运发唐金明
Owner SHENZHEN AUDAQUE DATA TECH
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