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Rapid molecular detection method and application for plasmopara viticola

A technology of grape downy mildew and a detection method, which is applied in the field of molecular detection of grape downy mildew, can solve the problems of being difficult to adapt to various detection environments, limited application and promotion, complicated operation procedures, etc., and achieves fast detection speed and improved efficiency. , the effect of easy and fast operation

Active Publication Date: 2013-08-14
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the above methods all have shortcomings such as long detection time, high equipment requirements, complicated operating procedures, and difficulty in adapting to various detection environments, which greatly limit their application and promotion in grassroots detection, and the above methods are limited by DNA extraction methods. , can not really meet the needs of "rapid" detection, so it is necessary to establish an accurate, efficient, fast, sensitive and economical detection method

Method used

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  • Rapid molecular detection method and application for plasmopara viticola
  • Rapid molecular detection method and application for plasmopara viticola
  • Rapid molecular detection method and application for plasmopara viticola

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Implementation example 1: Detection of downy mildew on the same grape variety in different regions of China

[0048] 1) Source of the strain

[0049] The strains were collected from grape growing areas in 15 provinces, 2 municipalities, and 3 autonomous regions in China (see Table 3), and the main grape variety currently cultivated in China——Red Earth was selected as the test target (the area without red earth cultivation is referred to as China The oldest breed - Kyoho instead).

[0050] The downy mildew bacterial strain of the same grape variety in different regions of China that the present invention adopts

[0051]

[0052] 2) Rapid extraction of strain DNA

[0053] Use a sterilized toothpick or tip to pick a small amount of mold layer on the surface of the sample, put it into a PCR tube, add 50 μL BufferA solution (100mM NaOH, 2% 20, Buffer A with 10M NaOH and 20% 20 freshly prepared), incubated at 95°C for 10 minutes. Then add 50 μL of BufferB solution (...

Embodiment 2

[0067] Implementation example 2: Detection of downy mildew in different grape varieties in the same area of ​​China

[0068] The strains were collected from Grape Grand View Garden in Tongzhou, Beijing, China, and the specific species information is shown in Table 4. The LAMP amplification reaction and procedure, and the identification of the LAMP product are the same as in Example 1.

[0069] The downy mildew bacterial strain of the different grape varieties in the same area of ​​China that the present invention adopts of table 4

[0070]

[0071] Implementation results

[0072] Utilize the LAMP primer combination of the present invention to use the DNA of different grape varieties Peronospora in the same area of ​​China as a template for LAMP amplification. The result is as Figure 5 , the PCR tubes numbered 1 to 17 under the ultraviolet light all showed green fluorescence, indicating that the samples contained different amounts of grape downy mildew DNA. The templates...

Embodiment 3

[0073] Implementation example 3: Detection of downy mildew in different grape varieties in different regions of China

[0074] The strains were collected from grape growing areas in 7 provinces, 2 municipalities, and 2 autonomous regions in China. There are 20 varieties in total. See Table 5 for specific information. The LAMP amplification reaction and procedure, and the identification of the LAMP product are the same as in Example 1.

[0075] The downy mildew bacterial strains of the different grape varieties in different regions of China that the present invention adopts

[0076]

[0077] Implementation results

[0078] The LAMP primer combination of the present invention is used to amplify the LAMP DNA of different grape varieties in different regions of China as templates. The result is as Image 6 , the PCR tubes numbered 1 to 27 under the ultraviolet light all showed green fluorescence, indicating that the samples contained different amounts of grape downy mildew D...

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Abstract

The invention belongs to the technical field of crop disease diagnosis and control, and in particular relates to a molecular detection method and application of plasmopara viticola. According to the molecular detection method, a group of looped-mediated isothermal amplification (LAMP) primers are self-designed through bioinformatic analysis according to the internal transcribed spacer (ITS) sequence information of a variety of disclosed peronosporales fungi and other common plant pathogenic fungi; and the existence of the deoxyribonucleic acid (DNA) of a plasmopara viticola genome at the level of 33fg / mu l can be rapidly detected from the plasmopara viticola samples of different varieties in different grape growing regions within only 55 minutes by combining a rapid extraction technology for the genome of the plasmopara viticola and adopting a one-step LAMP reaction. The rapid molecular detection method has high specificity, sensitivity and detection accuracy, and wide application range, and can be popularized and applied in various places as a rapid detection technology for the plasmopara viticola.

Description

(1) Technical field [0001] The present invention "a rapid molecular detection method and application of grape downy mildew", belongs to the technical field of crop disease diagnosis and prevention, and specifically relates to a molecular detection method and application of grape downy mildew. (2) Background technology [0002] Grape (Vitis vinifera) is one of the fruit trees with the largest cultivation area and the largest yield in the world. It has a cultivation history of 5,000 to 7,000 years. far more than other fruit trees. As a worldwide fruit, grapes have multiple values. With the improvement of people's living standards, the demand for pollution-free grapes and grape products is gradually increasing. It is the general trend for the grape industry to develop in a high-quality, green and healthy direction. Grape downy mildew has always been a major bottleneck restricting the grape and wine industry. It can reduce production by 10-20% in general years, and can reach 70...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 秦文韬张昊孔繁芳黄晓庆王忠跃
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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