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

A technology of tomato yellowing curved leaves and tomato koji, which is applied in the direction of biochemical equipment and methods, microbe measurement/inspection, etc., can solve problems such as difficult identification of viruses, and achieve accurate and reliable identification results, strong adaptability, and increased cost Effect

Active Publication Date: 2014-04-16
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 embodiment 1 primer

[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 For the genome sequences of leaf virus (TYLCGuV, International GenBank accession number AY602166) and Guangdong tomato leaf curl virus (ToLCGuV, International GenBank accession number AY602165), 4 pairs of specific primers were designed. figure 1 .

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

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

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

[0054] The primers used to detect Taiwan tomato leaf curl virus (ToLCTWV) were TWF and TWR, and the size of the PCR amplification product was 209bp;

[0055] T...

Embodiment 2

[0063] Example 2 Detection and Identification of Known Disease Samples

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

[0065] The flow chart of the method for detecting and identifying tomato yellow leaf curl disease caused by 4 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 put it in a 1.5 mL centrifuge tube; add 500 μL of tomato disease sample total DNA extraction buffer preheated at 65 °C, the buffer Components include 2% (w / v) CTAB, 1.4M NaCl, 0.02M EDTA, 0.01M Tris-HCl, add 0.2% (v / v) mercaptoethanol before use; place in 65°C water bath for 40 minutes; add 5...

Embodiment 3

[0074] Example 3 Detection and Identification Test of Unknown Disease Samples

[0075] A total of 10 tomato samples were collected from various regions in Guangdong. A total of 10 samples were obtained and numbered. The corresponding collection locations and symptoms of each sample are 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 with a single PCR (only 1 pair of primers among the above 4 pairs of primers are added to the single PCR reaction system, and the rest of the conditions are the same as in Example 2) , to be compared and verified.

[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 of Shantou, Zengcheng of Guangzhou, Gaoyao of Zhaoqing, Gaoyao of Zhaoqing, Huadu of Guangzhou, Huadu of Guangzhou, Sanshui of Foshan, Samples to be tested from Zengcheng, Guangzhou, Xinyi, Maoming, and Baiyun, G...

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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, in particular to tomato yellow leaf curl virus, Taiwan tomato leaf curl virus, Guangdong tomato yellow leaf curl virus and Guangdong tomato leaf curl virus, which can cause yellow leaf curl of tomato caused by infection alone or in combination. 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 preparing antibodies, preparing disease samples, coating detection plates, adding antibodies, adding labeled goat anti-rabbit antibodies, and color reaction. Although commercial antibodies to a few plant viruses can be purchased on the market, the vast majority of plant viru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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