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Kit and method for quantitatively testing tobacco mosaic virus

A technology for quantitative detection of tobacco mosaic virus, applied in the field of quantitative detection of tobacco mosaic virus, can solve the problems of low sensitivity, low accuracy, long detection time, etc.

Inactive Publication Date: 2017-04-26
WUXI FUYANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current detection methods have disadvantages such as low accuracy, low sensitivity, long detection time, professional skill requirements for the testers, and expensive professional instruments.

Method used

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  • Kit and method for quantitatively testing tobacco mosaic virus
  • Kit and method for quantitatively testing tobacco mosaic virus
  • Kit and method for quantitatively testing tobacco mosaic virus

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Effect test

preparation example Construction

[0048] In the present invention, the detection process is carried out in an antibody pre-coated microtiter plate, and the preparation method of the antibody pre-coated microtiter plate comprises the following steps:

[0049] 1) Dilute the specific mouse anti-tobacco mosaic virus monoclonal antibody with coating buffer to a concentration of 1.8-2.2 μg / ml, add it to the microtiter plate, react at 30-37°C for 2-5 hours or statically at 4-8°C overnight;

[0050] 2) washing the coated orifice plate with washing solution;

[0051] 3) Seal the washed orifice plate with blocking solution, and react at 30-37°C for 2-4 hours;

[0052] 4) drying the blocked orifice plate to obtain an antibody pre-coated microtiter plate.

[0053] In the present invention, the temperature of the first incubation and the second incubation is independently selected from 22-26° C., and the time of the first incubation and the second incubation is independently selected from 40-50 min.

[0054] In the prep...

Embodiment 1

[0087] Expression and Purification of Tobacco Mosaic Virus Protein

[0088] 1. Small-scale protein expression

[0089] (1) Vector construction

[0090] According to the protein sequence, the gene sequence was synthesized and constructed into the vector pET30a to obtain the pET30a-TMV positive plasmid.

[0091] (2) Strain activation: Tobacco mosaic virus positive plasmid was constructed using the commercial ET expression vector backbone sold by EMD Biosciences, transformed into BL21(DE3), and coated with LB solid medium with a kana concentration of 50 μg / mL. On the next day, pick a monoclonal colony and inoculate 5 mL of LB liquid medium with a kana concentration of 50 μg / mL, and incubate at 37°C for 12h to 14h.

[0092] (3) Small test expression: the next day, the inoculum of the strain was 2% by volume into 5 mL of LB liquid medium with a kana concentration of 50 μg / mL, and cultured at 37°C until OD 450 =0.4~0.6, draw 1 mL of bacterial solution and centrifuge it as the con...

Embodiment 2

[0103]Preparation of specific mouse anti-tobacco mosaic virus monoclonal antibody

[0104] 1. Monoclonal antibody preparation

[0105] (1) Immunogen preparation: The expressed and purified protein was mixed and emulsified with an equal volume of Freund's adjuvant as an antigen to form a water-in-oil state for immunization of mice.

[0106] (2) Immunization strategy: immunize 4 Balb / c mice with the protein, subcutaneously immunize 3 times with an interval of 4 weeks, and finally tested by ELISA, the antiserum titers are as follows:

[0107]

[0108] (3) Cell fusion: Two weeks after the last immunization, protein was injected intraperitoneally for booster immunization, and cell fusion was performed three days later. The mice were killed by neck dislocation, sterilized by soaking in 70% ethanol for 30 minutes, and the abdominal cavity was cut open on an ultra-clean table, the spleen was taken out, ground, passed through an 80-mesh sieve, and splenocytes were obtained, and SP2...

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Abstract

The invention provides a kit and a test method for quantitatively testing tobacco mosaic virus, which belong to the technical field of plant virus testing. The kit for quantitatively testing tobacco mosaic virus disclosed by the invention consists of following separately packaged reagents and an ELISA (Enzyme Linked Immunosorbent Assay) plate: the ELISA plate is a tobacco mosaic virus antibody-precoated ELISA plate; and the separately packaged reagents are: sample diluent, which is phosphate buffer; detergent, which is phosphate buffer containing 0.1 to 0.5 percent by weight of Tween-20; stop solution, which is H2SO4 solution with the concentration of 1.8mol / L to 2.2mol / L; enzyme-labeled antibody working solution, which is a horseradish peroxidase-labeled specific mouse anti-tobacco mosaic virus monoclonal antibody with the concentration of 2 mu g / ml; developer; and standard tobacco mosaic virus. The kit disclosed by the invention can implement the quantitative testing of tobacco mosaic virus, and is efficient, highly accurate and highly sensitive.

Description

technical field [0001] The invention relates to the technical field of plant virus detection, in particular to a kit and a detection method for quantitative detection of tobacco mosaic virus. Background technique [0002] Tobacco mosaic disease includes common tobacco mosaic disease and cucumber mosaic disease, which commonly occurs in various smoking areas in the world. Among them, there are also occurrences in the north and south smoking areas in my country, especially in the southern smoking areas. From the seedling stage to the harvest stage, the disease can occur in all tobacco areas, and the field strain incidence rate is 5% to 20%, and individual fields can be as high as 90% to 100%. After drying, the diseased leaves have uneven color, poor smoke smell and greatly reduced quality. The loss of the disease in the smoking area can reach 50-70%, or even loss of harvest. [0003] Tobacco mosaic virus (Tobacco mosaic virus, tobacco mosaic virus) is a single-stranded RNA ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/56983
Inventor 武爱波徐伟徐炜孙陈莉赵伟娟朱婕吴蔚
Owner WUXI FUYANG BIOTECH
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