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Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV)

A tomato yellowing curved leaf, accurate technology, applied in the direction of measuring device, color/spectral characteristic measurement, instrument, etc., to achieve the effect of high reliability, close detection accuracy rate, simple and fast operation

Inactive Publication Date: 2012-04-25
WEIFANG UNIV OF SCI & TECH +2
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Problems solved by technology

[0009] In order to solve the above problem that there is no quick and accurate method for detecting tomato yellow leaf curl virus, the present invention provides a popular, economical, practical and quick enzyme-linked Rapid and Accurate Detection of Tomato Yellow Leaf Curl Virus by Immunosorbent Assay

Method used

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  • Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV)
  • Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV)
  • Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV)

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Embodiment Construction

[0043] Used tool enzyme and chemical reagent source and preparation method are as follows:

[0044] xho I and Eco R I restriction endonuclease and alkaline phosphatase were purchased from Bao Biological Engineering Co., Ltd., Plant Genomic DNA Extraction Kit, Gel Rapid Purification Kit, Plasmid Small Extraction Kit, pEASY -T1 Simple cloning kit, protein quantification kit, etc. were purchased from Beijing Quanshijin Biotechnology Co., Ltd., protein purification column HiTrap Chelating HP (1 mL), antibody purification column HiTrap Protein A (1 mL) were purchased from GE Company of the United States . 2x Taq MasterMix, DNA Marker, and medium molecular weight standard protein were purchased from MBI Company; anti-His tag monoclonal antibody, HRP-labeled goat anti-rabbit IgG were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; IPTG, Freund's complete adjuvant (product number F5881 ), Freund's incomplete adjuvant (product number F5506), PNPP, etc. were purcha...

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Abstract

Relating to the technical field of virus detection in genetic engineering, the invention provides a method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV). The method comprises: cloning a TYLCV-CP gene segment from the total DNA of a diseased leaf, connecting the TYLCV-CP gene segment with pET-32a and transforming a recipient bacterium, conducting inducible expression to a positive bacterial colony and purifying the target protein; preparing antiserum with an immunized rabbit, labeling a purified antibody with alkaline phosphatase, and detecting a diseased plant to be detected by a DAS-ELISA (double antibody sandwich-enzyme-linked immunosorbent assay) method. The method for detection of TYLCV in the invention has high sensitivity and can be used for qualitative and quantitative analysis, with a lowest detection limit of 9.75ng / mL; it also has simple and rapid operation that can be finished within several hours; with strong specificity and good repeatability, the method has very obvious immunological reaction to TYLCV; characterized by high reliability, the detection accuracy of the method is very close to PCR (polymerase chain reaction) method; the detection cost is low, and the reagent has small consumption amount and can be stored for a long time. Thus, the method provided in the invention is very suitable for large scale routine detection.

Description

technical field [0001] The invention relates to the technical field of virus detection in genetic engineering, in particular to a method for rapidly and accurately detecting tomato yellow leaf curl virus. Background technique [0002] Tomato is one of the main vegetable varieties in my country. It is planted on a large scale in various vegetable producing areas across the country, and plays a very important role in the annual supply of vegetables. With the promotion of greenhouse vegetable planting technology and the improvement of economic benefits of off-season vegetable cultivation, the area of ​​tomato cultivation in greenhouses has increased year after year. At the same time, the occurrence of its diseases is also showing a trend of increasing gradually: tomato mosaic virus (Tomato mosaic virus, TMV) and cucumber mosaic virus (Cucumber mosaic virus, CMV) have been the main virus diseases in tomato production. However, in recent years, a new virus disease - tomato yel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N21/31
Inventor 乔宁李美芹竺晓平刘永光王兴翠国家进
Owner WEIFANG UNIV OF SCI & TECH
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