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Primers, kit and detection method for detecting bean yellow mosaic virus

The invention relates to a technology for a yellow mosaic leaf of common bean and a detection method, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., and can solve the problems of insufficient specificity, poor sensitivity, and low accuracy of detection results.

Active Publication Date: 2013-08-14
陈定虎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing primers used for RT-PCR detection of bean yellow mosaic virus are relatively not strong enough in specificity and relatively poor in sensitivity, which often leads to low accuracy of detection results

Method used

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  • Primers, kit and detection method for detecting bean yellow mosaic virus
  • Primers, kit and detection method for detecting bean yellow mosaic virus
  • Primers, kit and detection method for detecting bean yellow mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Kit of the present invention:

[0085] 1. Purpose:

[0086] Detection of BYMV in vegetable and flower plants by PCR method.

[0087] 2. Packing quantity:

[0088] 50 μl of RT-PCR reaction system for 50 times.

[0089] 3. Product Description

[0090] This kit adopts One Step RT-PCR method to detect BYMV in lily plants. This kit contains enzyme mix reagent, buffer, BYMV detection primers and positive control. The experimental operation is simple and convenient, preventing cross-contamination.

[0091] This kit has the following characteristics:

[0092] 3.1 Perform efficient RT-PCR amplification with a detection sensitivity of 0.1pg.

[0093] 3.2 The target product amplified by this kit has an A base attached to the 3' end, which can be directly cloned into T-Vector.

[0094] 4. Product content (50 times)

[0095]

[0096] 5. Storage: -20℃

[0097] 6. Detection method

[0098] 6.1 Prepare RT-PCR reaction solution according to the following composition.

[0...

Embodiment 2

[0110] RT-PCR of the present invention detects the detection method of bean yellow mosaic virus, comprises the following steps:

[0111] A. RNA extraction from the sample to be tested to obtain a template RNA solution;

[0112] a1. Select 0.1-0.2g of kidney bean leaf tissue showing suspicious symptoms, and use the same amount of healthy tissue as a control; add liquid nitrogen to freeze the lily leaf tissue, grind it thoroughly and put it into a 1.5mL centrifuge tube;

[0113] a2. Add 400 μL of ice-cold RNA extraction solution to the centrifuge tube, and mix evenly by inverting;

[0114] a3. Centrifuge at 12000g for 10min in a desktop refrigerated centrifuge at 4°C;

[0115] a4. Take the supernatant, add 400 μL of a mixture of phenol, chloroform, and isoamyl alcohol, wherein the volume ratio of phenol, chloroform, and isoamyl alcohol is 25:24:1, and mix evenly by inversion;

[0116] a5. Centrifuge at 12000g for 10min in a desktop refrigerated centrifuge at 4°C;

[0117] a6....

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Abstract

The invention discloses primers, a kit and a detection method for detecting a bean yellow mosaic virus. The sequences of the primers disclosed by the invention are BYMV Primer-1:5'-ccaacattccgccaaataat-3', and BYMV Primer-2:5'-aatacgaacaccaagcatgg-3'; and the kit and the detection method contain a primer BYMV Primer-1 and a BYMV Primer-2. The primers and the detection method for detecting the bean yellow mosaic virus disclosed by the invention 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 RT-PCR detection of bean yellow mosaic virus, belonging to the field of biotechnology. Background technique [0002] Bean yellow mosaic virus can infect a variety of plants such as legumes, Chenopodiaceae, amaranthaceae, and apricots, such as kidney beans, broad beans, cucumbers, tobacco, canna, gladiolus, etc.; the virus belongs to the family Potyviridae (Potyviridae), Potatovirus Y (Potyvirus) members. The length of virus particles is 700-750nmX13nm, the lethal temperature is 50-60°C, the dilution limit is 10-3-10-4, and the in vitro virus preservation period is 3-4 days. The main transmission media are: aphids, non-persistent transmission, which can be transmitted by mechanical inoculation. Bean yellow mosaic virus mainly infects the leaves, the diseased leaves turn yellow and distorted, the flower spikes grow difficult, and the flowers are few and small. [0003] The existing pr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/70C12N15/11C12R1/94
Inventor 陈定虎陈祖华王宏
Owner 陈定虎
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