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Rapid detection kit and method of strawberry mild yellow edge virus

A light yellow edge virus and kit technology, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effect of fast detection process and easy operation.

Active Publication Date: 2015-05-20
金禾佳农(北京)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other virus detection methods, reverse transcription loop-mediated isothermal amplification technology is fast, simple and sensitive. At present, there is no application of reverse transcription loop-mediated isothermal amplification technology in the detection of strawberry light yellow edge virus

Method used

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  • Rapid detection kit and method of strawberry mild yellow edge virus
  • Rapid detection kit and method of strawberry mild yellow edge virus
  • Rapid detection kit and method of strawberry mild yellow edge virus

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1, Strawberry mild yellow edge virus (Strawberry mild yellow edge virus) reverse transcription loop-mediated primer and its application

[0034] 1. Acquisition of reverse transcription loop-mediated primers for Strawberry mild yellow edge virus

[0035] According to the cDNA sequence of strawberry mild yellow edge virus (Strawberry mild yellow edge virus) RNA released by GenBank in NCBI of the United States (sequence accession numbers are AJ577339, AJ577359, D12515, D12517, EF143351, EU107085 and EU107086), through the software DNAMAN7.0 After homology analysis, the conserved sequence at the end of coat protein gene 3 of Strawberry mild yellow edge virus was found as a template, and the online software Primer3Input (http: / / bioinfo.ut.ee / primer3-0.4. 0 / primer3 / ) to design RT-LAMP primers, screen the designed primers, adjust the sequence, and verify, and finally obtain a set of LAMP primers with high sensitivity and specificity. The primers were synthesized by Sh...

Embodiment 2

[0063] Embodiment 2, kit of the present invention and application effect monitoring thereof

[0064] 1. Sensitivity detection of RT-LAMP method:

[0065] Carry out the mensuration of RT-LAMP and RT-PCR detection sensitivity according to the optimum technical scheme determined in embodiment 1, the object of detection is the RNA that extracts in the strawberry leaf that infects strawberry light yellow edge virus that selects in the above-mentioned reaction condition optimization process to obtain RNA stock solution.

[0066] The same RNA stock solution was diluted 10 times to obtain 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 and 10 7 Two times diluted dilutions were used as templates for RT-LAMP and RT-PCR amplification.

[0067] The RT-PCR amplification reaction mainly has two steps, the first step is the reverse transcription reaction: 10μM pd(N) 9 0.5 μl (Takara, Cat. No.: D3802), 10 μM Oligo dT 18 0.5 μl (Takara, Cat. No.: D511), 10 mM dNTP 0.5 μl, RNA 10 μl, add DEPC ddH ...

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Abstract

The invention discloses a special primer for detecting a strawberry mild yellow edge virus and an application thereof. The special primer for detecting the strawberry mild yellow edge virus is composed of a deoxyribonucleic acid (DNA) shown in a sequence 1 in a sequence table, a DNA shown in a sequence 2 in the sequence table, a DNA shown in a sequence 3 in the sequence table, and a DNA shown in a sequence 4 in the sequence table. The primer disclosed by the invention is utilized for detection, so that the method is rapid and sensitive in detection process, simple and convenient to operate, and free of other precious equipment and reagents, and is especially applicable to rapid detection in seedling breeding, detoxification cultivation and field investigation and grasping and application by basic technical personnel in production.

Description

technical field [0001] The invention relates to an RT-LAMP detection primer set, a detection kit and a method for strawberry light yellow edge virus. Background technique [0002] Strawberry mild yellow edge virus (SMYEV) is a single-molecule linear positive-sense ssRNA virus with a length of 5966 bp, belonging to the family Flexiviridae and the genus Potexvirus taxonomically. Virus particles are curved and linear, about 470-580nm long and 13nm wide. After SMYEV infects the host plant, it will exist in the epidermis and mesophyll cells of the host plant, and it is mainly transmitted by a variety of aphids in a persistent manner. The virus cannot replicate in the vector, nor can it be transmitted through the offspring of the vector; grafting can spread, Contact between seeds, pollen and plants does not spread. Strawberry mild yellow edge virus is usually asymptomatic after infecting Chilean strawberry (Fragaria chiloensis) and pineapple strawberry (Fragaria nilgerrensis), b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 尚巧霞陈柳崔高峰祝宁冉策陈笑瑜邢冬梅韩成贵魏艳敏赵晓燕杨瑞刘正坪张志勇
Owner 金禾佳农(北京)生物技术有限公司