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Method for rapidly and sensitively detecting tobacco potato virus Y

A sensitive detection, potato technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of poor specificity of serological detection methods, easy to be affected by external factors, high antibody price, etc., to overcome easy degradation And prone to false positives, saving detection time, and easy to degrade

Inactive Publication Date: 2017-07-21
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serological detection methods have poor specificity and are easily affected by external factors. The detection process takes a long time and the price of antibodies is high; traditional biological methods need to extract the total RNA of plants, and RNA is easily degraded during the extraction process

Method used

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  • Method for rapidly and sensitively detecting tobacco potato virus Y
  • Method for rapidly and sensitively detecting tobacco potato virus Y
  • Method for rapidly and sensitively detecting tobacco potato virus Y

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A method for rapid and sensitive detection of tobacco potato virus Y, comprising the steps of:

[0031] S1: To prepare the extract, take 0.5g of fresh leaves in a mortar, add 1ml of PBS to grind thoroughly, then transfer the grinding solution to a 2.0mL centrifuge tube, centrifuge at 12000rpm / min for 5min, and take the supernatant for later use;

[0032] S2: Take 5 μL supernatant and spot on 1 cm 2 On a polyvinylidene fluoride film (PVDF film) of the same size, place it in a fume hood to dry for about 10 minutes; then place 1cm 2 NCM into a 2.0mL centrifuge tube, add 100μLddHO 2 O, obtain sample 1 after sufficient vortex, then carry out RT-PCR reaction;

[0033] Among them, the method of reverse transcription is: take 2.0 μL of sample 1 and 3.0 μL of ddH 2 O was reacted at 95°C for 3 minutes, and then added 4.0 μL of 5×PCR buffer, 0.5 μL of 10 mM dNTPmix, 0.5 μL of RRI, 0.5 μL of PVY-R, 0.5 μL of M-MLV, and 9.0 μL of ddH 2 O was reacted at 42°C for 30 minutes;

[...

Embodiment 2

[0039] A method for rapid and sensitive detection of tobacco potato virus Y, comprising the steps of:

[0040] A1: To prepare the crude extract, take 0.5g of fresh tobacco leaves in a mortar, add 1mL of LPBS to thoroughly grind, transfer the grinding solution to a 2.0mL centrifuge tube, centrifuge at 12000rpm / min for 5min, and take the supernatant for later use;

[0041] A2: Use the above-mentioned crude extract as the template of the RT-PCR reaction system to perform RT-PCR reaction;

[0042] Among them, the method of reverse transcription is: take 2.0 μL of sample 1 and 3.0 μL of ddH 2 O was reacted at 95°C for 3 minutes, and then added 4.0 μL of 5×PCR buffer, 0.5 μL of 10 mM dNTPmix, 0.5 μL of RRI, 0.5 μL of PVY-R, 0.5 μL of M-MLV, and 9.0 μL of ddH 2 O was reacted at 42°C for 30 minutes;

[0043]The reaction method of PCR reaction is: take 2.5 μL of 10×Tag Buffer, 1.5 μL of dNTPmix, 1.5 μL of MgCl 2 , 0.5 μL of PVY-F, 0.5 μL of PVY-R, 0.25 μL of Ex Taq enzyme, 1.5 μL o...

Embodiment 3

[0048] A method for rapid and sensitive detection of tobacco potato virus Y, comprising the steps of:

[0049] B1: Extraction of plant total RNA,

[0050] First, weigh 0.1 g of plant material, quickly freeze it with liquid nitrogen, quickly grind it into a fine powder, transfer the fine powder to a 2.0 mL centrifuge tube, add 1 mL of Trizol to each tube, and obtain a lysate after standing for 5 minutes;

[0051] Then add 200 μL of chloroform to the above lysate, vortex to mix, place at 4°C for 5 min, and then centrifuge at 12000 rpm / min for 5 min at 4°C. After centrifugation, the liquid is divided into three layers, and the upper layer is absorbed solution into a 1.5mL centrifuge tube, add an equal volume of isopropanol, mix up and down, and stand at -20°C for 30-60min;

[0052] Then centrifuge at 12000rpm / min at 4°C for 15min, discard the supernatant, and collect the RNA pellet;

[0053] Then add 1mL of 75% ethanol to the above-mentioned tube for collecting the RNA precipit...

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Abstract

The invention discloses a method for rapidly and sensitively detecting a tobacco potato virus Y. A tobacco extracting solution is used as a reaction template, a polyvinylidene fluoride film is used as a reaction film, and an TR-PCR is combined to detect the potato virus Y, a tobacco mosaic virus and a cucumber mosaic virus. By adopting the method, the potato virus Y, the tobacco mosaic virus and the cucumber mosaic virus can be accurately and effectively detected, the operation is simple and convenient, the detecting sensitivity is improved, the detection time is shortened, and the detection cost is saved.

Description

technical field [0001] The invention relates to the technical field of plant protection, in particular to a method for rapidly and sensitively detecting tobacco potato virus Y. Background technique [0002] Tobacco virus disease is one of the important diseases of tobacco. At present, there are 16 kinds of tobacco virus diseases reported in my country, among which TMV, CMV, PVY and so on have caused serious losses. After tobacco is infected with viral diseases, the grade of tobacco leaves declines, and the quality deteriorates, which seriously hinders the development of tobacco production in my country. The prevention and treatment of tobacco viral diseases has become an urgent problem in production. [0003] At present, the detection methods for tobacco virus diseases mainly include indicator plant method, serological detection, molecular biology technology based on PCR, and isothermal ring-mediated technology. Serological detection methods have poor specificity and are ea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/6834
Inventor 孙现超代园凤朱虹李斌其他发明人请求不公开姓名
Owner SOUTHWEST UNIVERSITY
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