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Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease

A technology for tomato yellowing curved leaves and tomato koji, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as difficulty in identifying viruses, and achieve accurate and reliable identification results, strong adaptability, and increased cost. Effect

Active Publication Date: 2013-04-03
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

Since these virus infections all cause tomato yellowing and leaf curling symptoms, there is no significant difference, and field tomatoes are often infected by two or more viruses mixedly, it is necessary to determine which virus or several viruses are infecting a certain disease sample It takes more than 24 hours for detection and identification by any of the above two methods, and it is difficult to identify the virus species by serological detection methods

Method used

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  • Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease
  • Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease
  • Method and kit for synchronously detecting four viruses inducing tomato yellow leaf curl disease

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Experimental program
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Effect test

Embodiment 1

[0049] The design of the primer of embodiment 1

[0050] According to the four viruses that cause tomato yellow leaf curl disease, tomato yellow leaf curl virus (TYLCV, international GenBank accession number is JQ867092), Taiwan tomato leaf curl virus (ToLCTWV, international GenBank accession number is DQ237918), Guangdong tomato yellow leaf curl virus For the genome sequences of leaf virus (TYLCGuV, the international GenBank accession number is AY602166) and Guangdong tomato leaf curl virus (ToLCGuV, the international GenBank accession number is AY602165), four pairs of specific primers were designed. figure 1 .

[0051] The primers for detection of tomato yellow leaf curl virus (TYLCV) were TYF and TYR, and the PCR amplification product size was 321bp;

[0052] TYF: 5'-GACCTGGCCCCACATTGTTTTGCCT-3';

[0053] TYR: 5'-TCAGCAATCTGCCAACGACGCA-3';

[0054] The primers for the detection of Taiwan tomato leaf curl virus (ToLCTWV) were TWF and TWR, and the PCR amplification produc...

Embodiment 2

[0063] Example 2 Detection and identification of known disease samples

[0064] The known disease samples included 1 disease sample infected by TYLCV, ToLCTWV, TYLCGuV and ToLCGuV alone, 1 tomato disease sample infected by TYLCGuV and ToLCGuV mixed, and another healthy tomato sample was taken as a control.

[0065] The flow chart of the method for detection and identification of tomato yellow leaf curl caused by four kinds of virus infection is as follows: figure 2 shown.

[0066] (1) Extraction of total DNA from tomato samples

[0067] Use a sterile new blade to take 100 mg of leaf tissue from the leaves of the sample to be tested, grind it into powder with liquid nitrogen, and place it in a 1.5 mL centrifuge tube; add 500 μL of tomato disease sample total DNA extraction buffer preheated at 65°C. The ingredients include 2% (w / v) CTAB, 1.4M NaCl, 0.02M EDTA, 0.01M Tris-HCl, and 0.2% (v / v) mercaptoethanol before use; placed in a 65°C water bath for 40 minutes; added 500 μL o...

Embodiment 3

[0074] Embodiment 3 Detection and identification test of unknown disease samples

[0075] 10 tomato samples collected from many areas in Guangdong were sampled, a total of 10 samples were obtained and numbered, and the corresponding collection locations and symptoms of each sample were shown in Table 1. The method used for detection and identification is the same as that used in Example 2, and each sample is detected by a single PCR (only one pair of primers in the above 4 pairs of primers are added to the single PCR reaction system, and the rest conditions are the same as those in Example 2). , to compare and verify.

[0076] The result is as Figure 4 and as shown in Table 1, Figure 4 Middle: M: standard molecular weight (100bp gradient); 1-10: Chenghai, Shantou, Zengcheng, Guangzhou, Gaoyao, Zhaoqing, Gaoyao, Guangzhou, Huadu, Guangzhou, Sanshui, Foshan, respectively. Samples to be tested from Zengcheng in Guangzhou, Xinyi in Maoming and Baiyun in Guangzhou; 11: Standar...

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Abstract

The invention discloses a method and a kit for synchronously detecting four viruses inducing a tomato yellow leaf curl disease and belongs to the field of plant protection. The four viruses include tomato yellow leaf curl virus, Taiwan tomato leaf curl virus, Guangdong tomato yellow leaf curl virus and Guangdong tomato leaf curl virus. Four pairs of specific primers are designed; total DNA of a sample which is to be detected is extracted; the extracted DNA is taken as a template and the four pairs of specific primers are taken as primers to perform PCR (polymerase chain reaction) in the same system; the PCR product is detected; and according to the fragment size of the PCR product, the virus which invades the sample can be identified. The method and the kit are quick, simple, convenient, accurate and low in cost, can be used for synchronously detecting the four viruses which induce the tomato yellow leaf curl disease, and can be applied to the actual production to quickly, accurately and effectively detect the plant disease sample.

Description

technical field [0001] The invention belongs to the field of plant protection, and particularly relates to four viruses, namely tomato yellow leaf curl virus, Taiwan tomato leaf curl virus, Guangdong tomato yellow leaf curl virus and Guangdong tomato leaf curl virus, which cause tomato yellow leaf curl caused by individual or mixed infection. Rapid detection methods and kits for diseases. Background technique [0002] For the detection and identification of plant viruses such as tomato yellow leaf curl virus, there are mainly two existing technologies: serological detection methods and polymerase chain reaction (PCR) detection and identification methods. [0003] The serological detection method mainly includes the steps of antibody preparation, disease sample preparation, coating detection plate, adding antibody, adding labeled goat anti-rabbit antibody, and color reaction. Although a few commercial antibodies to plant viruses can be purchased on the market, most of the an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 何自福杜振国汤亚飞佘小漫
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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