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Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method

A lily mottle virus, detection kit technology, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem that there is no specific detection kit for LMoV, which is expensive and difficult Meet the needs of detoxification detection of flowers and edible lilies, and achieve good market prospects, high sensitivity and good specificity

Active Publication Date: 2010-12-15
NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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Problems solved by technology

[0009] At present, there is no commercialized LMoV immunoassay kit at home and abroad. The well-known plant virus kit detection company Agdia has only commercialized the general immunological test strip of the genus Potatovirus Y, and there is no kit for the specific detection of LMoV. Foreign plant virus diagnostic reagents are expensive. For example, Agdia’s kits are mostly between RMB 1,500 and 4,000. It is difficult to meet the needs of domestic flowers and edible lilies for virus-free detection.

Method used

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  • Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method
  • Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method
  • Lily mottle virus double-antibody sandwich enzyme-linked immunosorbent assay kit and preparation method

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Embodiment 1

[0060] 1. Preparation method of LMoV recombinant fusion protein PI200:

[0061] 1) Extraction of total RNA from leaves of diseased lily plants by Trizol method:

[0062] A. Weigh 40 mg of leaves of lily diseased plants, put them into a mortar, and grind them into powder quickly in liquid nitrogen. After the liquid nitrogen was volatilized, 1 mL of Trizol (Invitrogen Company) was added, transferred into a 1.5 mL centrifuge tube, and allowed to stand at room temperature for 5 minutes.

[0063] B. Add 200 μL of chloroform, mix up and down several times, and place at room temperature for 2 to 3 minutes. Centrifuge at 12,000 g for 10 min at 4°C.

[0064] C. Take the upper aqueous phase, add 500 μL of isopropanol, mix well and let stand at -20°C for 20 minutes, then centrifuge at 12,000g for 10 minutes at 4°C.

[0065] D. Discard the supernatant, wash the pellet with 1 mL of 75% ethanol, and centrifuge at 7,500 g for 5 min at 4°C.

[0066] E. Air-dry the precipitate and dissolve...

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Abstract

The invention relates to a lily mottle virus (LMoV) double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit and a preparation method. The assay kit comprises a 96 micropore ELISA plate, an enzyme labeled antibody, a buffer solution, a developing solution and a stop solution, wherein the 96 micropore ELISA plate is coated with an LMoV specific polyclonal antibody, and the enzyme labeled antibody is an LMoV specific polyclonal antibody labeled by horse radish peroxidase. An immunogen used in the invention is a fusion recombinant antigen of LMoV coat protein and cytoplasmic inclusion protein, which is expressed by genetic engineering. The double-antibody sandwich enzyme-linked immunosorbent assay established by antibodies produced by the immunization can be used for detecting lily leaf specimens. Compared with other detection methods, the double-antibody sandwich enzyme-linked immunosorbent assay has high detection sensitivity and high accuracy, and ng-level viral antigenscan be detected. The preparation method of the kit is simple, convenient, quick, economical and practical.

Description

technical field [0001] The invention relates to a biotechnology product used for plant virus detection, in particular to a lily mottle virus (LMoV) double-antibody sandwich enzyme-linked immunoassay (DAS-ELISA) detection reagent which can be used for rapid and sensitive detection of lily mottle virus disease Kit, the present invention also relates to the preparation method of the kit. Background technique [0002] Lily (Lilium spp.) is traditionally used as a medicinal and edible plant, and has become one of the most important cut flower ornamental flowers in the world. The Netherlands is the world's largest producer of lily bulbs. In 2005, the production area of ​​Dutch lily bulbs reached 3800hm 2 , accounting for about 72% of the total production area of ​​cut lily in the world. Cut flower lily occupies the fourth place in the output value of my country's flower industry. At the same time, lily is also an important economic crop as food and medicine. With the growing ar...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535C12N15/40C12N15/63C07K19/00C07K16/10
Inventor 童勋章谢忠奎王亚军张玉宝安丽萍郭志鸿张亚娟
Owner NORTHWEST INST OF ECO-ENVIRONMENT & RESOURCES CAS
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