Immuno comb array test paper for detecting antibody of simian MeV (measles virus) as well as preparation method and application

A technology for detecting test strips and viruses, which is applied in the directions of viruses/bacteriophages, biochemical equipment and methods, and biological testing, etc. The effect of the experiment cycle

Inactive Publication Date: 2016-06-08
海南出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Once an outbreak occurs, it spreads quickly and is difficult to control
Measles virus is a stable, monotypic virus that is difficult to eradicate completely, interfering with experimental studies

Method used

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  • Immuno comb array test paper for detecting antibody of simian MeV (measles virus) as well as preparation method and application
  • Immuno comb array test paper for detecting antibody of simian MeV (measles virus) as well as preparation method and application
  • Immuno comb array test paper for detecting antibody of simian MeV (measles virus) as well as preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Design and synthesis of embodiment 1SOE-PCR primers

[0061] According to the gene sequence of simian MeV virus, SOE-PCR amplification primers were designed using the principle of overlap extension PCR (SOE-PCR). The primer sequences are as follows:

[0062] MeVF: 5'-GAATTCTGATCAAAGTGAGAATGAGCTCCCAAGTGATCAAAA

[0063] GTGAGAATGAGCTCCCAA-3'

[0064] MeVR1:5'-GAGCTCATTCTCACTTTGATCACTTGGGAGCTCATTCTCA-3'

[0065] MeVR2: 5'-CTCGAGCTTGGGAGCTCATTCTCACTTTGATCACTTGG

[0066] GAGCTCATTCTCACTTTGATC-3'

Embodiment 2

[0067] Cloning and sequencing of the simian MeV virus MeV32 gene of embodiment 2

[0068] (1) Cloning of simian MeV virus MeV32 gene

[0069] Carry out SOE-PCR with the primers designed and synthesized in Example 1, and the PCR reaction system is as follows:

[0070] The first round of PCR reaction conditions:

[0071] The mixture was pre-denatured at 95 °C for 5 min, denatured at 94 °C for 1 min, annealed at 60 °C for 30 s, extended at 72 °C for 1 min, 15 cycles, with a total extension of 10 min at 72 °C.

[0072] The above mixture is as follows:

[0073] MeVF primer

1 μL

MeVR primers

0.5 μL

2×Taq MasterMix

15 μL

Ultra-pure water

Make up to 30 μL

[0074] The second round of PCR reaction conditions:

[0075] The mixture was pre-denatured at 95 °C for 5 min, denatured at 94 °C for 1 min, annealed at 63 °C for 30 s, extended at 72 °C for 1 min, 30 cycles, and a total extension of 10 min at 72 °C.

[0076] The above mixture...

Embodiment 3

[0083] The construction of embodiment 3 expression vector

[0084] pMD18T-MeV and pGEX-4T-1 were digested with EcoRI and XhoI respectively, and the digested target fragments were gel recovered. Connect the recovered target fragments, the connection system is as follows:

[0085] target segment

5 μL

pGEX-4T-1

2 μL

T4 DNA Ligase

1 μL

10×Ligase Buffer

1 μL

wxya 2 o

1 μL

[0086] Ligate overnight at 16°C.

[0087] The ligation product was transformed into BL21(DE3)pLysS competent cells, the positive colonies were screened by ampicillin resistance, and the plasmid was extracted for identification by PvuⅠ digestion. The result is as figure 2 shown. The positively identified plasmid was named pGEX-4T-MeV.

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Abstract

The invention mainly relates to immuno comb array test paper for detecting an antibody of a simian MeV (measles virus) as well as a preparation method and an application. A recombinant plasmid pGEX-4T-MeV for establishment of an immuno comb array is shown in the sequence table Seq No.4. An upper primer MeVF in a primer pair for establishment of the recombinant plasmid is shown in the sequence table Seq No.1, a lower primer MeVR1 of the primer pair is shown in the sequence table Seq No.2, and a lower primer MeVR2 of the primer pair is shown in the sequence table Seq No.3. The establishment method comprises the following steps: the SOE (splicing overlapping extension)-PCR (polymerase chain reaction) primer pair is designed according to the nucleotide sequence of the simian MeV, simian MeV genes are amplified through SOE-PCR, and the recombinant plasmid pGEX-4T-MeV is established; finally, the immuno comb array for quickly detecting the antibody in blood or serum of an experimental monkey is prepared with a film chromatographic technique according to the enzyme linked immunosorbent assay principle and has good stability, specificity, sensitivity and repeatability.

Description

technical field [0001] The invention relates to a detection method in the field of biotechnology and the technical field of antigens, in particular to an immune comb detection method for monkey MeV virus antibody and a detection kit thereof. Background technique [0002] Measles is an acute infectious disease of humans and other primates caused by measles virus (Measles virus, MeV), and it is one of the diseases that must be strictly controlled in monkey farms. Once an outbreak occurs, its spread is fast and difficult to control. Measles virus is a stable monotype virus, which is difficult to eliminate completely, which interferes with experimental research. In recent years, the number of experimental monkeys in my country has grown rapidly, with a total of more than 200,000. The import and export trade volume of experimental monkeys is increasing year by year. With the increasing requirements for the quality of experimental monkeys in the international market, measles vir...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/70C12N15/66G01N33/68G01N33/569G01N33/52
CPCC12N15/11C12N15/66C12N15/70C12N2800/101G01N33/52G01N33/56983G01N33/68G01N2333/12
Inventor 李丹丹徐义刚高慎阳王昱
Owner 海南出入境检验检疫局检验检疫技术中心
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