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Method for detecting five plant viruses synchronously

A technology for simultaneous detection of plant viruses, applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of less research and application of multiplex PCR technology, low accuracy and sensitivity, and long time consumption

Inactive Publication Date: 2014-01-29
SOUTHWEST UNIV
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Problems solved by technology

Since Chamberlain et al. first reported the use of multiplex PCR to diagnose Duchenne muscular dystrophy (DMD) in 1988, there have been a large number of literature reports on the detection of diseases in animals, especially humans, and in plant pathology research, the use of multiplex PCR technology relatively few research applications
At present, the commonly used plant virus detection method in production is ELISA, which is not very accurate and sensitive.
Moreover, studies have found that compound virus infections on field plants are common and complex. If the conventional single RT-PCR technique needs repeated experiments to detect various viruses one by one, it will not only take a long time, but also be costly.
The multiplex RT-PCR detection system of the above five common plant viruses has not been reported

Method used

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  • Method for detecting five plant viruses synchronously
  • Method for detecting five plant viruses synchronously

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Embodiment Construction

[0032] Get 6 plant samples such as Chinese cabbage, tobacco, tomato, potato, capsicum and pumpkin, detect by the method of the present invention, the result is as follows: figure 2 shown. The type of virus infected by each sample can be clearly determined from the figure, which proves the feasibility and accuracy of the method of the present invention for simultaneous detection of five plant viruses.

[0033] Specific steps:

[0034] (1) Extraction of total RNA

[0035] Take 0.1g fresh leaves from 6 plant samples respectively, according to the kit (RNAiso TM Plus, TaKaRa) method to extract total RNA.

[0036] (2) Multiplex RT-PCR

[0037] Using the extracted total RNA as a template, the first-strand cDNA was synthesized using a reverse transcription kit (PrimeScript RT reagent Kit, TaKaRa Company). The reverse transcription system was 10 μL: 2.5 μL total RNA, 2 μL 5×PrimeScript Buffer, 0.5 μL Oligo dT Primer, 0.5 μL Random 6 mers, 0.5 μL PrimeScript RT Enzyme Mix I, 4 μ...

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Abstract

The invention relates to a method for detecting five plant viruses synchronously, wherein the method comprises the following steps: extracting plant total RNA, preparing a cDNA (complementary Deoxyribose Nucleic Acid) template through reverse transcriptional reaction, performing multiplex PCR (Polymerase Chain Reaction) reaction, and determining species of viruses through electrophoresis detection. By designing specific primers of the five viruses and optimizing the primer concentration and PCR reaction conditions, five plant viruses can be detected synchronously. The method for detecting five plant viruses synchronously is fast and accurate, avoids the repeatability of the general PCR detection, is good in detection stability and strong in specificity, and is suitable for quickly detecting the plant samples so as to guide crop production and reduce disease losses.

Description

technical field [0001] The invention belongs to the technical field of plant virus detection, in particular to a method for synchronous detection of five plant viruses. Background technique [0002] Plant virus is an important pathogen, and the number of diseases caused by it accounts for the second place in plant diseases. It can cause a variety of serious virus diseases in plants all over the world. There are many kinds of viruses that infect plants, among them, tobacco mosaic virus ( Tobacco mosaic virus , TMV), cucumber mosaic virus ( Cucumber mosaic virus , CMV), turnip mosaic virus ( Turnip mosaic virus , TuMV), Potato virus Y ( Potato virus Y , PVY) and Potato virus X ( Potato virus X , PVX) is the main type of virus that damages vegetables, tobacco and fruit trees, and the damage is serious. Taking cucumber mosaic virus (CMV) as an example, it can infect more than 1,000 species of plants including monocotyledonous and dicotyledonous plants, and is one of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2531/113
Inventor 杨会房青玲王春艳刘陈晨孙现超
Owner SOUTHWEST UNIV
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