Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method

A technology of tomato yellowing curved leaves and bemisia tabaci, applied in the biological field, can solve the problems that serological methods cannot be applied to the detection of vector insects, the inability to clarify the poisoning situation of single bemisia tabaci, and strong dependence on instruments, etc., to achieve Reliable detection results, low cost and high sensitivity

Active Publication Date: 2014-07-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, the detection of bemisia tabaci infection rate could only rely on the PCR method, but this method can only detect mixed samples of multiple bemisia tabaci due to the limitations of strong instrument dependence and high cost, so the experimental data obtained by the PCR method is only a rough value, it is impossible to determine the poisonous situation of a single Bemisia tabaci
In addition, due to the essential difference between plants and insects, serological methods suitable for plants cannot be applied to the detection of vector insects

Method used

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  • Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method
  • Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method
  • Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Detection of poisonous rate of Bemisia tabaci samples in the field

[0058] The present invention utilizes the method of dot-ELISA to detect the carrying situation of tomato yellow leaf curl virus by whitefly in the field, calculates the virus carrying rate of whitefly, predicts the outbreak trend of local tomato yellow leaf curl virus, and takes preventive measures in advance , to serve agricultural production.

[0059] 110 Bemisia tabaci were collected from the field of the Vegetable Institute of Hangzhou Academy of Agricultural Sciences with an insect extractor ( figure 1 ) and stored in a 4°C refrigerator for a short period of time. After removing Bemisia tabaci from the refrigerator at 4°C, place it in a petri dish and wash it with ddH 2 O soaking, dipped the tip of a gun to pick up a single head of Bemisia tabaci, placed it on a clean parafilm, and dried it at room temperature. Draw a line with a pencil on the outer paper of the nitrocellulose membr...

Embodiment 2

[0060] Example 2: Development of a dot-ELISA kit for detecting tomato yellow leaf curl virus carried by Bemisia tabaci

[0061] 1. Detection of tomato yellow leaf curl virus (TYLCV) dot-ELISA kit ( Figure 4 ) contains the following materials and reagents:

[0062] 1) Kit instruction manual 1 copy

[0063] 2) Reagents and materials:

[0064] 0.05 mol / L PBS 10 ml

[0065] TYLCV monoclonal antibody 0.1 ml

[0066] HRP-labeled goat anti-mouse IgG secondary antibody (A4416 Sigma) 0.1 ml

[0067] TMB chromogenic substrate solution (W4121 Promega) 10 ml

[0068] 10× concentrated blocking solution (0.1 mol / L PBST) 10 ml

[0069] 10× Concentrated Antibody Diluent (50% skimmed milk powder, 0.1mol / L PBST) 20 ml

[0070] 20× concentrated washing solution (0.2 mol / L PBST) 60 ml

[0071] Blocking agent (skimmed milk powder) 15 g

[0072] NC film (Amersham) 5 sheets

[0073] 2. The kit instruction manual includes:

[0074] 2.1 Principle of the kit:

[0075] dot-ELISA is an ELISA...

Embodiment 3

[0110] Example 3: Using a kit to detect Bemisia tabaci in the field

[0111] The kit can be used to detect the virus-carrying rate of Bemisia tabaci samples in the field, and thousands of Bemisia tabaci can be detected at the same time. The results are fast, reliable, high-throughput and easy to operate. It is used to investigate the situation of tomato yellow leaf curl virus carried by Bemisia tabaci on a large scale in the field, and to predict the outbreak of the disease in time. Figure 6 It is part of the results of using the kit to detect Bemisia tabaci in Hangzhou field vegetables.

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Abstract

The invention discloses a dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying a tomato yellow leaf curl virus and application of the method. Field bemisia tabaci is collected, pretreated and grinded, monoclonal antibody of the tomato yellow leaf curl virus is used, the dot-ELISA method which is used for detecting virus transmission medium bemisia tabaci is established, a quick kit is developed and researched, and the tomato yellow leaf curl virus which is carried by the bemisia tabaci is detected. By the aid of the method, the virus carrying rate of the field bemisia tabaci can be surveyed in a large scale, and the field tomato yellow leaf curl disease outbreak trend is predicted. The method is quick, efficient, sensitive, special, high in flux and simple to operate. The tomato yellow leaf curl disease can be diagnosed quickly, predicted and controlled scientifically.

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 Bemisia tabaci carrying tomato yellow leaf curl virus. Background technique [0002] Tomato yellow leaf curl virus (TYLCV) belongs to Geminiviridae ( Geminiviridae ) Begomovirus ( Begomovirus ), the genus virus can only be infected by Bemisia tabaci under natural conditions ( Bemisia tabaci ) is transmitted in a persistent manner, so it is also known as whitefly-borne geminivirus. The large-scale epidemic of tomato yellow leaf curl disease is often accompanied by the outbreak of the vector, Bemisia tabaci. In my country, tomato yellow leaf curl disease was initially only distributed in Hainan, Yunnan, Guangdong and Guangxi provinces. Since the discovery of tomato yellow leaf curl virus on tomatoes in Shanghai and Zhejiang in 2006, the disease has spread rapidly in East China. Since then, it has become popular o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
Inventor 谢艳周雪平吴建祥矫晓阳刘欢
Owner ZHEJIANG UNIV
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