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LAMP technology-based primer, kit and detection method for detecting Plum pox virus

A plum pox virus, technical detection technology, applied in the biological field, can solve the problems of easy degradation, false positive, cumbersome RNA extraction, etc.

Inactive Publication Date: 2016-06-15
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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  • LAMP technology-based primer, kit and detection method for detecting Plum pox virus
  • LAMP technology-based primer, kit and detection method for detecting Plum pox virus
  • LAMP technology-based primer, kit and detection method for detecting Plum pox virus

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Effect test

Embodiment 1

[0112] Kit of the present invention:

[0113] 1. Product specification: 50 times

[0114] 2. Features:

[0115] Loop-mediated isothermal amplification method loop-mediated isothermal amplification method, LAMP is a new gene amplification method, which uses only one enzyme to amplify at a constant temperature, and uses 6 primers that can recognize 6 specific sequences of the target DNA, so the specificity High, and the amplification efficiency is very high, it can be amplified in a short time, and the reaction result can be judged visually.

[0116] 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. The sample to be tested and the reagents in this kit need to be mixed according to the requirements, and then placed at a certain temperature at 63°C for about 1 hour to react, and the result can be judged from the amplifi...

Embodiment 2

[0140] The present invention detects the detection method of plum pox virus, comprises the following steps:

[0141] A. Using nano magnetic beads to extract the RNA of the sample to be tested;

[0142] The immune nanomagnetic beads are produced by the following process:

[0143] ① Preparation of nanomagnetic beads

[0144] Take 5.2gFeCl 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 resulting precipitate 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 25mLFe 3 o 4 Magnetic nanoparticle absolute ethanol solution, then diluted with absolute ethanol to 150mL, ultrasonically osc...

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Abstract

The invention discloses primers, a kit and a detection method for detecting plum pox virus based on LAMP technology. The primers include: forward outer primer F3: 5'-CCTTTGATTTCTACGAGATGAC-3'; reverse outer primer B3: 5'-ACCACTACACTCCCCTC-3 '; forward internal primer FIP: 5'-TCCAAGCCAAATAAACGATTTTGAAGTGAGGCACATATTCAAATGAAGG-3'; wherein said forward internal primer FIP comprises F1c and F2; reverse internal primer BIP, wherein said reverse internal primer BIP comprises B1c and B2; loop Primers: LF: 5'-CATTTCTCAATGCTGCTG-3'; LB: 5'-ACCGCTGGTGATGTTAA-3'. The purpose of the present invention is to overcome the deficiencies in the prior art, to provide a quick, easy, efficient, high accuracy, strong specificity, high sensitivity, primers, kits, Method for detection of plum pox virus.

Description

technical field [0001] The invention relates to a primer, a kit and a detection method for detecting plum pox virus based on LAMP technology, and belongs to the field of biotechnology. Background technique [0002] Plumpox virus, PPV is one of the most dangerous viruses in drupe fruit trees. Once the fruit tree is infected by it, it will cause a large number of unripe fruits to fall off, severely reduce the yield, and greatly reduce the quality of the fruit. The speed of transmission is fast, and it may bring catastrophic losses to the development of the fruit tree industry of the whole country in a short period of time. The virus was first discovered in Bulgaria in 1915, and then quickly spread to most countries in Europe, as well as the Mediterranean, Central Africa, India and Chile, and has also been reported in the United States and Canada. Because of the seriousness of the virus, it has been listed as a quarantine pathogen of type A2 by the European Plant Protection Or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6806C12Q1/6844
Inventor 陈定虎杨雷亮管维杨翠云邓丛良王振华
Owner 陈定虎
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