Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof

A technology of reovirus and pseudo-cave blue crab is applied in the direction of analysis materials, measuring devices, instruments, etc., which can solve the unfavorable use and daily monitoring of rural areas and grassroots units. There is no effective treatment for the disease, and economic losses are inevitable. and other problems, to achieve the effect of simple operation and control, high stability and fast detection of test paper

Active Publication Date: 2016-02-10
ZHEJIANG WANLI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the diagnosis of the disease is based on the observation of the symptoms. When the symptoms appear, the disease has already occurred, and there is no effective treatment for the disease, and its economic loss is inevitable.
The second is to directly observe the virus in the tissue with an electron microscope after negatively staining the ultra-thin section of the animal tissue, which requires expensive instruments and skilled technicians who have undergone strict training. or imminent symptoms, e

Method used

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  • Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof
  • Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof
  • Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof

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Embodiment 1

[0044] As shown in the accompanying drawing, the immune colloidal gold test paper of the present invention capable of detecting the pseudocave blue crab reovirus, the test paper is composed of sample pad 1, binding pad 2, nitrocellulose membrane 3, absorption pad 4, hard polychloride The vinyl liner 5 is formed by bonding and combining into one body, wherein, the middle part of the nitrocellulose membrane 3 is spray-coated with a detection line 6 and a quality control line 7, and the colloidal gold bonding pad 2 is made of glass cellulose membrane, and the colloidal gold bonding Colloidal gold-labeled anti-scylla reovirus p29 protein monoclonal antibody 1M9 (G-1M9) was sprayed on the pad 2, and the monoclonal antibody was immunized with purified His-p29 protein to Balb / c mice, through cell fusion, Secreted by the screened hybridoma cell line 1M9; the nitrocellulose NC membrane is the detection membrane of the test strip, the upper left is the detection line 6, the quality contr...

Embodiment 2

[0045] Example 2 Preparation of Recombinant Antigen Against Scylla reovirus p29 Protein

[0046] Using specific primers, the 12th segment (S12) of the viral genome corresponding to the p29 protein of Scylla reovirus was amplified by polymerase chain reaction, and the open reading frame (ORF) of the SsRVS12 gene was successfully amplified , the S12 gene ORF was cloned into the Escherichia coli expression vector pET28a(+), and the prokaryotic recombinant expression vector pET28-S12 was constructed:

[0047] Forward primer: 5'-CGGGATCCATGAACCTGGAAATTAACAAC-3' (SEQ ID NO.1)

[0048] Reverse primer: 5'-CCCAAGCTTAGTAATCGAGAACCCACTT-3' (SEQ ID NO.2)

[0049] The recombinant vector PMD18-S12 containing the full-length segment of reovirus S12 was used as a template for PCR amplification. The 25 μL reaction system contained: 1 μL of plasmid PMD18-S12 (100 ng / μL), 1 μL of upstream and downstream primers (20 μM), dNTPmix (10mM) 1μL, 10×PCRBuffer 2.5μL, TaqDNA polymerase 0.5μL, deionized...

Embodiment 3

[0052] Example 3 Preparation of Monoclonal Antibody 1M9 Against Scylla reovirus p29 Protein

[0053] Using Qiagen's QIAexpressionist TM The protein purification system purifies the p29 protein of the histidine tag (His) recombinantly expressed in Escherichia coli (the recombinant protein is referred to as: His-p29); mix the purified His-p29 protein with an equal amount of Freund's complete adjuvant, and inject subcutaneously for immunization BALB / c mice. Three weeks later, the expressed and purified His-p29 protein mixed with an equal amount of Freund's incomplete adjuvant was injected intraperitoneally. After 3 weeks, the rats were boosted with His-p29 protein once. Three days after the third immunization, the spleen was killed by anesthesia, and the spleen cells were fused with non-secretory myeloma cells (SP2 / 0), and the fused cells were continuously cultured with RPMI1640 solution containing HAT and HT. The culture medium containing HAT and HT was screened, and the anti...

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Abstract

The invention relates to a Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and belongs to the field of biological detection. SSRV recombinant antigen p29 expression, purification, monoclonal antibody preparation, polyclonal antibody preparation, colloidal gold immunochromatographic assay reagent strip preparation and sensitivity and specificity of the Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip on detecting SSRV antigens are included. The invention further relates to a preparing method and a using method of the Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip. Compared with traditional virus extracting, grinding, centrifuging and the like, the colloidal gold immunochromatographic assay reagent strip established through the method has the advantages of being convenient and fast to use, sensitive, special and the like, a definite diagnosis result can be obtained within 10 minutes, and the colloidal gold immunochromatographic assay reagent strip is especially suitable for clinical use and use in grassroots units.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a colloidal gold immunochromatographic reagent strip for Scylla pseudocaveus reovirus, a preparation method thereof and an application in detection of Scylla pseudocaveus reovirus. Background technique [0002] Scyllaserratareovirus (SsRV) is one of the infectious pathogens that cause serious harm in the breeding industry of Scyllas Scyllas (hereinafter referred to as Scyllas). The blue crab farming industry has caused huge economic losses. At present, due to the unclear background of the epidemic and the slow progress in the research and development of control technology, it often appears powerless to control the disease. Therefore, early detection of SsRV virus infection to prevent the spread of the virus has become the primary means of prevention and control of "clear water disease". To discover the SsRV virus-infected animals to the greatest extent, it is nec...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/577
CPCG01N33/558G01N33/56983G01N33/577
Inventor 陈吉刚刘静雯范东洋徐苗苗袁洋洋杨季芳
Owner ZHEJIANG WANLI UNIV
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