Method for detecting aphid or plant infection RhPV virus

A detection method and aphid technology, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem of lack of rapid molecular biology detection methods, shorten the time required for detection, reduce the sample volume, and achieve accurate detection. The effect of increasing the degree of

Inactive Publication Date: 2008-04-30
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

At present, the detection of RhPV virus mainly adopts the method of enzyme-linked detection and immunohistochemical localization, and there is no related rapid molecular biological detection method

Method used

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  • Method for detecting aphid or plant infection RhPV virus
  • Method for detecting aphid or plant infection RhPV virus

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Experimental program
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Effect test

Embodiment 1

[0012] Embodiment 1, the detection method of RhPV virus infection in aphids or plants of the present invention detects RhPV virus infection in aphids

[0013] 1. Extraction of aphid sample RNA: randomly collect 10 aphids from the susceptible aphid (Rhopalosiphumpadi) population (aphid population cultivated with plants carrying the RhPV virus) respectively, divide into 2 groups, and 5 of each group are used as samples to be tested. RNeasy Plant Minikit Kit (Qiagen) was used to extract total RNA from the tested samples. The specific steps are as follows: Grind the live aphids obtained from each group in 450 μl RLT buffer solution, mix them and transfer them to a QIAshredder spin column (lilac) column (Qiagen), centrifuge at 13,000 rpm, and collect the supernatant after 2 minutes , add 1 / 2 volume of ethanol (volume percentage 96-100%) in the supernatant, mix immediately, transfer to RNeasy mini column (Qiagen), centrifuge, 10,000rpm, 15s, discard the lower layer of centrifugate, ...

Embodiment 2

[0019] Embodiment 2, the detection method of aphid or plant infection RhPV virus of the present invention detects RhPV virus infection in a single aphid

[0020] Extraction of single head sample RNA: Randomly collect single head aphids from the susceptible aphid (Rhopalosiphum padi) population (aphid population cultivated with plants carrying the RhPV virus), collect 5 aphids in total and divide them into 5 groups, with 1 head in each group, Total RNA was extracted from single aphid samples using RNeasy Plant Minikit kit (Qiagen). Specifically: put each live aphid obtained in 450 μl RLT buffer to grind, mix well and transfer to QIAshredder spin column (lilac) column (Qiagen), centrifuge at 13,000 rpm, collect supernatant after 2 min solution, add 1 / 2 volume of ethanol (96-100% by volume) in the supernatant, mix immediately, transfer to RNeasy mini column (Qiagen), centrifuge, 10,000rpm, 15s, discard the lower centrifugate, Add 500μl RPE buffer to the RNeasy mini column, centr...

Embodiment 3

[0022] Embodiment 3, the detection method of aphid or plant infection RhPV virus of the present invention detects RhPV virus infection in plants

[0023] 1. Extraction of plant sample RNA: wheat plants randomly damaged by aphids, cut leaves 0.5-1cm 2 , take 3 samples (one sample for each wheat plant). Take a 0.5-1cm leaf of a wheat plant that is harmed by poisonous aphids. 2 As a positive control, a wheat leaf 0.5-1cm without poisonous aphids 2Used as a negative control. The sample to be tested, the positive control and the negative control were taken, and the total RNA of the sample to be tested was extracted using the RNeasy Plant Minikit kit (Qiagen). Specifically: grind the obtained leaves in 450 μl RLT buffer, mix well and transfer to a QIAshredder spin column (lilac) column (Qiagen), centrifuge at 13,000 rpm, collect the supernatant after 2 minutes, and add the supernatant 1 / 2 volume of ethanol (96-100% by volume), mix immediately, transfer to RNeasy mini column (Qia...

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Abstract

The invention discloses an aphid or plant infected RhPV virus detecting method. The method is realized by that the total RNA of the aphid or plant tissue sample is extracted, and the RT-PCR reaction is performed through the specificity primer pair of a nucleotide sequence as the sequence 1 in a sequence table and the nucleotide sequence as the sequence 2 in the sequence table, and if the nucleotide sequence obtained through the detection amplification is the nucleotide sequence of the 8316-8689th positions of the (5) end with the GENBANK number of AF022937, the aphid or the plant is infected with the RhPV virus. Through the RT-PCR method, the method of the invention can detect the distribution and the existence of the RhPV in the aphid and the host plant quickly, efficiently and accurately; the required detection time is greatly shortened, only 4 to 5 hours are required from sample gathering to the detection accomplishment, the sample quantity required in the detection is greatly reduced, and simultaneously the detection accuracy is greatly enhanced.

Description

technical field [0001] The invention relates to a detection method for RhPV virus infection of aphids or plants. Background technique [0002] RhPV is an insect virus that specifically infects aphids and can control the population of aphids. In the past, the detection of RhPV virus was mainly carried out by enzyme-linked detection (ELISA) or by electronmicroscopical methods. These methods are time-consuming and have low sensitivity and accuracy, especially it is difficult to detect the presence and distribution of RhPV in aphid host plants. [0003] Molecular biology techniques have been increasingly applied to research in many fields including virology. RT-PCR has been used in virology research as a molecular biological technique, but there is no report on its application to RhPV virus detection. At present, the detection of RhPV virus mainly adopts the method of enzyme-linked detection and immunohistochemical localization, and there is no related rapid molecular biologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 班丽萍曲鲁江
Owner CHINA AGRI UNIV
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