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Immuno comb array test paper for detecting antibody of SIV (simian immunodeficiency virus) as well as preparation method and application
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A technology for detecting test strips and viruses, which can be used in viruses/phages, biochemical equipment and methods, and biological testing.
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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Problems solved by technology
Although foreign kits have high accuracy, they are expensive. If a large-scale SIV serological survey is carried out, domestic experimental monkey breeding units cannot afford it. Therefore, they often only purchase a small amount of foreign kits for one-time testing of experimental monkeys that are about to be exported.
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Embodiment 1
[0056] Design and synthesis of embodiment 1SOE-PCR primers
[0057] According to the gene sequence of monkey SIV virus, SOE-PCR amplification primers were designed using the principle of overlap extension PCR (SOE-PCR). The primer sequences are as follows:
[0062] Cloning and sequencing of the simian SIV virus SIV30 gene of embodiment 2
[0063] (1) Cloning of SIV30 gene of monkey SIV virus
[0064] Carry out SOE-PCR with the primers designed and synthesized in Example 1, and the PCR reaction system is as follows:
[0065] 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.
[0066] The above mixture is as follows:
[0067] SIVF primers
2 μL
SIVR primers
2 μL
2×Taq MasterMix
15 μL
Ultra-pure water
Make up to 30 μL
[0068] PCR amplification results were detected by agarosegel electrophoresis, such as figure 1 As shown, it shows that the experiment obtained specific DNA fragments consistent with expectations.
[0074] pMD18T-SIV 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:
[0075] target segment
5 μL
pGEX-4T-1
2 μL
T4 DNALigase
1 μL
10×Ligase Buffer
1 μL
wxya 2 o
1 μL
[0076] Ligate overnight at 16°C.
[0077] 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 SmaⅠ and EcoRI double enzymedigestion identification. The result is as figure 2 shown. The positively identified plasmid was named pGEX-4T-SIV.
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Abstract
The invention mainly relates to immuno comb array test paper for detecting an antibody of an SIV (simian immunodeficiency virus) as well as a preparation method and an application. A recombinant plasmid pGEX-4T-SIV for establishment of an immuno comb array is shown in the sequence table Seq No.3. An upper primer SIVF in a primer pair for establishment of the recombinant plasmid is shown in the sequence table Seq No.1, and a lower primer SIVR of the primer pair is shown in the sequence table Seq No.2. The establishment method comprises the following steps: the SOE (splicing overlapping extension)-PCR (polymerasechain reaction) primer pair is designed according to the nucleotide sequence of the SIV, SIV genes are amplified through SOE-PCR, and the recombinant plasmid pGEX-4T-SIV 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 field of antigen technology, in particular to a test paper for immunocomb detection of monkey SIV virusantibody, a preparation method and application thereof. Background technique [0002] Simian Immunodeficiency Virus (SIV) is an exogenous retrovirus that is potentially infectious to humans. SIV infection breeding monkey population will directly affect its application in biomedical research. Therefore, the quarantine requirements of the importing country and the exporting country of the experimental monkeys clearly stipulate that the experimental monkeys must be negative for SIV antibodies. At present, except for a few laboratories such as BioReliance and VRL in the United States that have experimental monkey SIV ELISA kits, there is no similar product on the market. Although foreign kits have high accuracy, they are expensive. If a large-scale SIV serological survey ...
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