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Primer and kit as well as method for detecting cymbidium mosaic virus

A detection method, a technology for building orchid leaves, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the cumbersome RNA extraction, limit the rapid, simple and efficient detection of plant virus disease samples , false positives, etc.

Active Publication Date: 2015-04-22
陈定虎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-efficiency antiserum must be available for serological reactions. In addition, serological testing cannot accurately measure some samples with low virus content and interfering substances, and false positive results are prone to occur, and there is a problem of long detection time. The problem is that it cannot meet the requirements of rapid detection at the current port; RT-PCR technology is one of the most widely used new technologies in molecular biology. Compared with ELISA detection, the detection of viruses by RT-PCR technology has the characteristics of high sensitivity and can be detected. Minimum virus content to reach fg level
At present, many new technologies are continuously derived on the basis of RT-PCR technology, such as real-time fluorescent RT-PCR technology, gene chip technology, etc., so that the scope of application is continuously expanded, and the specificity and sensitivity are also continuously improved. However, these molecular biological However, RNA extraction is cumbersome and easy to degrade, and PCR reaction is easily interfered by polyphenols and polysaccharides in the host, which limits the rapid, simple and efficient detection of plant virus disease samples

Method used

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  • Primer and kit as well as method for detecting cymbidium mosaic virus
  • Primer and kit as well as method for detecting cymbidium mosaic virus
  • Primer and kit as well as method for detecting cymbidium mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Kit of the present invention:

[0116] 1. Product specification: 45 times

[0117] 2. Features:

[0118] Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) is a new type of gene amplification method, which uses only one enzyme to amplify at a constant temperature, and uses 4 primers that can recognize 6 specific sequences of the target DNA , so the specificity is high, and the amplification efficiency is very high, and a large amount of amplification can be performed in a short time, and the reaction result can be judged visually.

[0119] This kit adopts the loop-mediated isothermal amplification method, and uses ribonucleic acid (RNA) as a template to directly carry out a simple kit for reverse transcription loop-mediated isothermal amplification. This kit uses an enzyme solution mixed with reverse transcriptase and DNA polymerase , you only need to mix the sample to be tested with the reagents in this kit according to the requirem...

Embodiment 2

[0148] The present invention detects the detection method of Jianlan mosaic virus, comprises the following steps:

[0149] A. Using nano magnetic beads to extract the RNA of the sample to be tested;

[0150] The immune nanomagnetic beads are produced by the following process:

[0151] ① Preparation of nanomagnetic beads

[0152] Take 5.2g FeCl respectively 3 ·6H 2 O, 2.7g FeSO 4 ·7H 2 O. Dissolve 0.85mL of 12.1moL / L concentrated hydrochloric acid in 200mL of water, ultrasonically deoxygenate, and then drop the above solution into 250mL, 0.75moL / L NaOH solution; stir at 80°C, after the reaction is over, use a magnetic separator to separate the obtained The precipitate was separated from the reaction medium, washed 3 times with deionized water and 2 times with absolute ethanol, and made into 5g / mL Fe 3 o 4 Magnetic nanoparticles absolute ethanol solution; take 25mL Fe 3 o 4 Magnetic nanoparticle absolute ethanol solution, then diluted with absolute ethanol to 150mL, ult...

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Abstract

The invention discloses a primer and a kit as well as a method, which are used for detecting a cymbidium mosaic virus. The primer comprises a forward outer primer F3: 5'-CAGTTTTGCGCTTACTACGC-3', a reverse outer primer B3: 5'-GGCGCCGTACTTCCCTAT-3', a forward inner primer FIP:5'-ACCAGCCTTGGCCCAGTTG-AGTGGTGTGGAATCTGATGC-3' and a reverse inner primer BIP: 5'-TCGATGCCGTTGATTCCACTGC-CACGTTCACGGTCAGTAGG-3'. The kit and the detection method comprise the primer. The primer and the detection method are used for detecting the cymbidium mosaic virus rapidly, simply, conveniently and efficiently and are high in accuracy, strong in specificity and high in sensitivity.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for detecting Jianlan mosaic virus, belonging to the field of biotechnology. Background technique [0002] Orchid is a world-renowned flower and is deeply loved by people all over the world. Hundreds of millions of orchid seedlings enter and exit my country every year. However, orchids are very easy to carry viruses, especially Cymbidium mosaic virus ( CymMV) and Odontoglossum ringspot virus (ORSV), and detoxification is still very difficult, which seriously restricts the sustainable and healthy development of my country's orchid industry and the constraints of foreign technical barriers. Therefore, the only way to effectively strengthen the import and export Quarantine of plant viruses, continuous research and development of new detection methods, continuous improvement of high-precision detection of viruses carried by inbound and outbound orchid seedlings, and early elimination of virus pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 陈定虎陈祖华王宏曹小茂杨雷亮彭志民刘恭源王勇朱春红
Owner 陈定虎
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