Kit for rapidly detecting potato viruses Y and detection method

A potato and kit technology, which is applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of limiting the popularization and application of RT-PCR detection method, difficult and rapid detection of RT-PCR detection method, etc. Highly sensitive detection, shortened preparation time, and programmable effects

Inactive Publication Date: 2011-08-03
TOBACCO RES INST CHIN AGRI SCI ACAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because conventional PCR detection requires expensive PCR machines, gel electrophoresis and imaging systems, it is difficult to use RT-PCR detection method for rapid on-site detection, which greatly limits the popularization and application of RT-PCR detection method in production

Method used

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  • Kit for rapidly detecting potato viruses Y and detection method
  • Kit for rapidly detecting potato viruses Y and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 The detection kit of the present invention consists of the following components (taking the packaging that can detect 4 samples as an example, it can also be designed to detect multiple samples according to this principle):

[0037] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0038] (2) Rinsing tubes, 6 pieces, 1ml of sterilized distilled water is housed in each tube;

[0039] (3) Nucleic acid denaturation tubes, 6 pieces, each containing 20 μL of TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0);

[0040] (4) Amplification detection tubes, 6 pieces, each containing 24 μL of amplification reaction solution and 1 μL of dye. The upstream primer 2 and downstream primer 2 of the primers are each 0.2μM, each of dATP, dTTP, dGTP and dCTP is 1.6mM, MgCl 2 7mM, Betaine (betaine) 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 2mM, (NH 4 ) 2 SO 4 10mM, Triton X-100 0.1% (mass volume ratio), reverse transcriptase 5U, Bst DNA ...

Embodiment 2

[0048] Embodiment 2 The detection kit of the present invention can also be made up of following parts (can detect the packing of 4 samples):

[0049] (1) Sampling tubes, 4 pieces, used to hold and grind samples to be tested;

[0050] (2) Rinsing tubes, 6 pieces, each tube is equipped with 1ml of distilled water;

[0051] (3) Nucleic acid denaturation tubes, 6 pieces, each tube is filled with 20 μL of TE buffer solution (containing 10 mM Tris-HCl and 1 mM EDTA pH8.0);

[0052] (4) Amplification detection tubes, 6 pieces, each tube is equipped with 25 μL of amplification reaction solution and 1 μL of nucleic acid dye, the components of the amplification reaction solution are as follows: each of the upstream primer 1 and downstream primer 1 of the amplification primers is 1.6 μM, 0.2 μM each of upstream primer 2 and downstream primer 2 of the amplification primers, 1.4 mM each of dATP, dTTP, dGTP, and dCTP, MgCl 2 8mM, Betaine (betaine) 1.2M, Tris-HCl 20mM, KCl 10mM, MgSO 4 ...

Embodiment 3

[0059] Embodiment 3 Use the detection kit of the present invention to detect the detection effect experiment of potato virus Y

[0060] (1) Take about 0.1 g of the sample tissue of potato virus Y infection and non-infection respectively and place it in a sampling tube, and quickly grind the sample to a slurry state with a grinding rod;

[0061] (2) Dip the slurry sample with a grinding rod to fully wet the FTA diaphragm;

[0062] (3) Draw the quick-drying liquid onto the above-mentioned FTA membrane, and let the FTA membrane stand at room temperature for 10 minutes;

[0063] (4) Use a toothpick to transfer the above-mentioned FTA membrane that has adsorbed the sample nucleic acid to the rinse tube, and simultaneously place the FTA membrane without the nucleic acid of the potato virus Y (negative control) and the FTA membrane that has absorbed the nucleic acid of the potato virus Y (positive control) Transfer to the rinsing tube, shake the rinsing tube vigorously for 3 minutes...

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Abstract

The invention relates to the field of virus detection, in particular to a kit for rapidly detecting potato viruses Y (PVY for short) and a detection method thereof. The rapid detection kit comprises a sampling tube, a rinsing tube, a nucleic acid denaturing tube, an amplifying and detecting tube, a negative control tube, a positive control tube, a rapid drying solution as well as a FTA (Fault Tree Analysis) membrane, a grinding bar, toothpicks, a suction tube and the like which are respectively packaged in a sterile bag. The formed detection method simplifies operation steps, reduces experimental cost and has stronger practicality in fields or rooms.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a rapid and highly sensitive detection kit for potato virus Y (PVY for short) and a detection method thereof. Background technique [0002] The genome of Potato virus Y is a single RNA positive strand, and its mitochondria are slightly curved and linear, about 680-900nm long and 11-12nm wide. Potato virus Y has been prevalent in Europe since 1953, especially in areas where potatoes are widely planted. It expanded in the Americas in the 1970s, and occurred in the tobacco areas of Northeast China, Huanghuai and Southwest China. If infected with the vein necrosis strain of potato virus Y within 4 weeks of tobacco transplanting, it can lead to complete failure of production and harvest. The color and luster and smoke smell of the diseased leaves are poor after drying, and their quality is greatly reduced. Therefore, it is particularly urgent and important to develop new technologies ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 刘艳华王志德戴培刚张兴伟任民罗成刚牟建民
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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