A kind of detection method of apple mosaic disease

A technology of apple mosaic virus and detection method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of difficulty in taking into account virus mutation strains, heavy workload, high cost, etc., and expand the range of detection objects , easy to observe, easy to judge the effect

Active Publication Date: 2022-07-15
北京市林业果树科学研究院
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Problems solved by technology

However, due to the common phenomenon of multi-virus co-infection on apple plants in the field, conventional detection methods can only detect a single virus or even a single virus strain. Detecting multiple viruses and virus strains one by one is not only labor-intensive and costly , and it is difficult to take into account the characteristics of many virus mutant strains, which may easily cause missed detection (ie false negative)

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  • A kind of detection method of apple mosaic disease
  • A kind of detection method of apple mosaic disease
  • A kind of detection method of apple mosaic disease

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of a detection method for apple mosaic disease

[0032] 1.1 Main reagents

[0033] EASYspin Plant RNA Extraction Kit (Beijing Biomed Technology Development Co., Ltd.), Reverse Transcription cDNA Synthesis Kit (Bao Bioengineering Dalian Co., Ltd.), SYBR Reaction Solution (Bao Bioengineering Dalian Co., Ltd.), primer synthesis (Beijing Qingqing Co., Ltd.) Kexinye Biotechnology Co., Ltd.), cloning vector pMD19-T and other conventional reagents (Beijing Leichuang Biotechnology Co., Ltd.).

[0034] 1.2 Real-time fluorescent quantitative PCR reaction

[0035] (1) Extract the total RNA of the apple leaf, branch, flower or fruit sample to be tested, and reverse-transcribe it into cDNA;

[0036] (2) Reaction system: 5.0 μL of SYBR reaction solution, 0.5 μL of 10pM / μL primer solution of SEQ ID No.1 and 0.5 μL of SEQ ID No.2 primer solution, 1.0 μL of cDNA template, and 3.0 μL of RNase-free Water, a total of 10 μL;

[0037] (3) Reaction conditions: The r...

Embodiment 2

[0049] Example 2 Field application of apple mosaic disease detection

[0050] 2.1 Detection object

[0051] In June 2018, a total of 13 apple varieties or rootstock leaves in the apple orchards of the headquarters and experimental base of Beijing Forestry and Fruit Tree Research Institute were collected, and the method of the present invention was applied to detect apple mosaic disease.

[0052] In March 2019, high-temperature detoxification was performed on the above-mentioned apple varieties or rootstock mosaic disease-positive plants; from May to August 2019, the method of the present invention was applied to each detoxified seedling plant obtained by grafting of detoxified young shoots. Initial inspection and re-inspection of leaf disease.

[0053] 2.2 Test results

[0054] The test of 13 apple varieties or rootstocks in the field showed that apple plants with many years of age, such as Fuji, Gala, Wanglin, Hong Jonagold, and rootstock SH6, were positive regardless of wh...

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Abstract

The invention discloses an efficient and rapid detection method for apple mosaic disease, which can detect whether apple plants carry Prunus necrotic ring spot virus (PNRSV), apple necrotic mosaic virus (ApNMV) and apple mosaic virus (ApMV) at one time 3 viruses. Through the design and optimization of specific degenerate primers, the cDNA of apple leaves, branches, flowers or fruit samples is used as the template to carry out real-time quantitative PCR amplification reaction, and the specific dissolution curve is analyzed to determine whether it carries apple mosaic disease, with high compatibility and high compatibility. High sensitivity features. The detection method of the invention has simple operation and good stability, and is suitable for rapid detection of apple samples collected in the field, thereby providing guidance for the formulation of prevention and control measures for apple mosaic diseases and reducing losses caused by diseases.

Description

technical field [0001] The invention relates to the field of plant protection and molecular biology detection, in particular to a detection method for apple mosaic disease. Background technique [0002] my country is a big apple producing country, and its planting area and total output have ranked first in the world for many years. However, viral diseases are common in apple-producing areas such as Shaanxi, Shandong and Beijing in my country. The combined infection rate of different viral diseases is over 90%, resulting in a 20-30% reduction in yield and a 25% reduction in commercial fruit rate. Virus diseases have become a serious impact on fruit products. One of the main factors of yield and quality. Apple trees are poisoned for life after being infected by the virus, and there is currently no effective drug to remove them. The virus proliferates in the fruit tree, disturbing and destroying the normal physiological function of the tree, resulting in decreased growth, decr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2527/107
Inventor 李兴亮张军科李民吉周贝贝张强魏钦平
Owner 北京市林业果树科学研究院
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