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Rapid detection test strip for epizootic hemorrhagic disease virus antibody and preparation method of rapid detection test strip

A deer epidemic hemorrhage and antibody detection technology, which is applied in the field of animal virology and zoonosis, can solve the problems of EHDV introduction, and achieve the effects of low detection cost, small amount of detection samples, good specificity and sensitivity

Active Publication Date: 2014-03-19
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there is no report of EHD prevalence in my country, EHD has been reported in many countries and regions, such as the United States, Australia, South America and some countries in Africa. With the rapid development of international animal and animal product trade, there are foreign countries and regions. The risk of EHDV being introduced into my country

Method used

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  • Rapid detection test strip for epizootic hemorrhagic disease virus antibody and preparation method of rapid detection test strip
  • Rapid detection test strip for epizootic hemorrhagic disease virus antibody and preparation method of rapid detection test strip
  • Rapid detection test strip for epizootic hemorrhagic disease virus antibody and preparation method of rapid detection test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Expression of truncated EHDVVP7 protein

[0027] 1. EHDV VP7 protein epitope analysis and codon optimization: by searching the NCBI database of EHDV VP7 gene sequence and corresponding amino acid sequence (AY351653), using online software to analyze its main epitope, confirm that the selected epitope is located in VP7 The 97-304th amino acid in the protein, its amino acid sequence is SEQ ID No. 1 in the sequence list, and its corresponding nucleotide sequence is the 289-912 nt nucleotide (AY351653).

[0028] 2. Codon optimization and artificial synthesis of EHDV VP7 gene: Codon optimization, that is, redesign the gene sequence according to the preference of the expression system for amino acid codons, and modify the low or rare codons in the gene sequence for expression The system uses frequent codons to ensure that the amino acid sequence of the expressed protein remains unchanged. According to E. coli’s preference for codons, the selected gene sequence (SEQ ID ...

Embodiment 2

[0043] Example 2: Preparation of colloidal gold-labeled test strips

[0044] 1. Preparation of colloidal gold and labeling of colloidal gold SPG: The colloidal gold solution was prepared by the reduction method of trisodium citrate. Add 1mL of 1% chloroauric acid solution to 99mL of tri-distilled water, heat to boiling, quickly add 2.4mL of freshly prepared 1.05% (m / v) trisodium citrate solution, and mix well, continue heating for about 5-10 minutes, the solution The color can be changed from blue to red. After cooling to room temperature, filter with 0.22μm 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 minutes, slowly add 10% ovalbumin (OVA) to its final concentration of 10 mg / mL, and continue stirring for 30 minutes. Centrifuge at 4,000r / min for 10min, transfer the supernat...

Embodiment 3

[0050] Example 3: Test of specificity, sensitivity and coincidence rate of test strips

[0051] 1. The specificity of the test strip: use the test strip to detect standard positive serum samples of EHDV (type 1-8), and other related viruses including Akabane disease virus (AKV), foot-and-mouth disease virus type O (FMDV O), blue tongue Positive serum samples and negative control samples of BTV, Petit Peste des Ruminants (PPRV) and Bovine Viral Diarrhea Virus (BVDV). The results showed that the test strip was positive when detecting EHDV (type 1-8) standard positive serum samples, and it was negative when detecting other virus positive serum and negative control samples, indicating that the test strip has good specificity.

[0052] 2. Sensitivity of the test strip: Use the test strip to detect 5 serially diluted EHDV positive serum samples (numbered 1 to 5), and use the ELISA kit as a control at the same time. The result shows that the test strip detects 5 sample antibody drops The...

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Abstract

The invention discloses a rapid detection test strip for an epizootic hemorrhagic disease virus (EHDV) antibody and a preparation method of the rapid detection test strip. The rapid detection test strip mainly adopts a truncated EHDV VP7 expression antigen as a detection line reagent. Antigen epitope software is used for analyzing EHDV VP7 and a segment containing a main antigen epitope is selected to optimize a codon corresponding to the segment; a DNA (Deoxyribonucleic Acid) segment is artificially synthesized according to an optimized nucleotide sequence and a recombinant expression carrier is constructed; colon bacillus is transferred to carry out protein expression to obtain a truncated EHDV VP7 recombinant antigen; the truncated EHDV VP7 recombinant antigen is purified and then is used for detecting the EHDV antibody. The rapid detection test strip established based on the truncated EHDV VP7 expression antigen has the advantages of convenience for operation, rapidness in detection, no need of special laboratories and equipment and the like; the limitation of an existing detection method is overcome; the rapid detection test strip can be used for rapid detection on the EHDV antibody and blood serum epidemiological investigation.

Description

Technical field [0001] The invention relates to the fields of animal virology and animal infectious diseases, in particular to a rapid detection test strip for detecting deer epidemic hemorrhagic disease virus antibodies and a preparation method thereof. Background technique [0002] Epizootic hemorrhagic disease of deer (EHD) is caused by epizootic hemorrhagic disease of deer virus (EHDV), which causes white-tailed deer body temperature rise, mucosal and serous surface extensive bleeding and frequent A severely fatal infectious disease characterized by death in a coma. Cattle and sheep can also be infected, seriously affecting the development of animal husbandry and international trade in many countries. EHD is one of the important insect-borne diseases, and Culicoides is the main transmission vector. The World Organization for Animal Health (OIE) lists EHD as a notifiable infectious disease. Although there is no report of EHD epidemic in my country, because EHD has been repor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C12N15/70
CPCG01N33/56983G01N33/6878G01N2333/14
Inventor 杨俊兴孙洁连慧香陈兵花群义吕建强曹琛福张彩虹卢体康胡运发秦智锋刘建利
Owner SHENZHEN AUDAQUE DATA TECH
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