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Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application

A tomato spotted wilt virus and serological technology, applied in the field of rapid detection of thrips, can solve the problems of no reports and achieve the effect of simple operation, high accuracy and low cost

Active Publication Date: 2015-04-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no serological method for the detection of TSWV in thrips has been reported so far.

Method used

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  • Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application
  • Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application
  • Serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1: detection of thrips sample poisoning rate

[0090] Collect 160 thrips reared in the laboratory, dip a single thrips with a brush, place them on a clean parafilm, add 2 ul of 0.05 mol / L PBS dropwise to each head, and grind them with the bottom of a 0.5 ml centrifuge tube. Use a pipette gun to draw 1-2 μL of thrips homogenate, spot on the center of the nitrocellulose membrane, and set positive and negative controls. Let the membrane stand to dry, add about 10 ml of 5% skim milk powder, and block at 37°C for 30 minutes; discard the skim milk powder, add 1:3000 times diluted monoclonal antibody against tomato spotted wilt virus, and incubate at 37°C for 1 hour ; Discard the liquid, shake and elute with PBST (0.01 mol / L PBS containing 0.05% Tween-20) 3 times, 3 minutes each time; discard the eluate, add 1:2000 diluted horseradish peroxidase (HRP )-labeled secondary antibody (A4416 Sigma), and incubated at 37°C for 1 hour; discard the liquid, shake and elute wi...

Embodiment 2

[0091] Example 2: Development of a dot-ELISA kit for detection of tomato spotted wilt virus carried by thrips

[0092] 1. Detection of thrips carrying tomato spotted wilt virus (TSWV) dot-ELISA kit ( figure 2 ) contains the following materials and reagents:

[0093] 1) Kit instruction manual 1 copy

[0094] 2) Reagents and materials:

[0095] 0.05 mol / L PBS 10ml

[0096] TSWV monoclonal antibody 0.1 ml

[0097] Positive control (poisonous thrips homogenate) 0.1 ml

[0098] Negative control (non-toxic thrips homogenate) 0.1 ml

[0099] HRP-labeled goat anti-mouse secondary antibody 0.1 ml

[0100] TMB chromogenic substrate solution 10 ml

[0101] 10× Concentrated Blocking Solution 10 ml

[0102] 10× Concentrated Antibody Diluent 20 ml

[0103]20× Concentrated Wash Solution 60 ml

[0104] Sealant 15 g

[0105] NC film 5 pieces

[0106] 2. The kit instruction manual includes:

[0107] 2.1 Principle of the kit:

[0108] dot-ELISA is an ELISA detection method based o...

Embodiment 3

[0132] Example 3: Using a kit to detect the rate of thrips in the field

[0133] The kit can be used to detect the virus-carrying rate of thrips samples in the field, and hundreds of thrips can be detected at the same time. The results are fast, reliable, high-throughput and easy to operate, which provides technical support for early prediction of the occurrence and prevalence of the disease. image 3 It is part of the results of using the kit to detect Thrips yunnanensis. The test results showed that 26 of the 258 thrips were positive ( image 3 ), the calculated rate of thrips to TSWV is 10.08%, and it is predicted that there will be an epidemic trend of TSWV in this area. It is recommended to control thrips with pesticides to prevent the prevalence and outbreak of TSWV.

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Abstract

The invention discloses a serological method for rapid detection of tomato spotted wilt virus carried by individual thrips and its application A hybridoma cell strain 1A5By capable of secreting monoclonal antibody against tomato spotted wilt virus and having a preservation number of CGMCC No.9343 is utilized to establish a dot-ELISA method for detection of tomato spotted wilt virus carried by individual thrips, to research and develop a rapid detection kit, and to rapidly detect the virus carried rate of the field thrips carrying tomato spotted wilt virus. The invention can be used in mass rapid investigation of virus carried rate of the thrips and in early prediction of tendency and outbreak of the tomato spotted wilt virus. The detection method is fast, efficient, sensitive and specific, and has high throughput and easy operation, and provides technical support for the rapid diagnosis, prediction, forecast and scientific prevention and control of the tomato spotted wilt virus disease.

Description

technical field [0001] The field of the invention belongs to the field of biotechnology, and in particular relates to a rapid detection method and application of thrips carrying tomato spotted wilt virus. Background technique [0002] Tomato spotted wilt virus (TSWV) was first discovered in Australian tomatoes in 1919, and now it occurs in tropical, subtropical, and temperate regions of the world, and is widely distributed in Europe, North America, South America, Asia, Oceania, etc. countries. TSWV has a wide range of hosts and can infect 160 species of dicotyledonous plants and 10 species of monocotyledonous plants, causing serious economic losses to agricultural production. TSWV leads to the formation of small black spots on the leaves of susceptible plants, purple and brown necrotic streaks along the veins on the back of the leaves, dwarfing of diseased plants or wilting leaves, chlorotic ring spots and brown necrotic spots on immature fruits, and ring spots on matur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/56983G01N33/577G01N2333/08
Inventor 谢艳吴建祥周雪平刘雪建
Owner ZHEJIANG UNIV
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