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Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction)

A technology of RT-PCR and step method, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of many influencing factors, increase test pollution and manual misoperation, and reduce the impact Factors, reducing sample cross-contamination, shortening the effect of detection time

Active Publication Date: 2013-02-13
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The two reaction steps and stages correspond to two different reaction systems, and there are many influencing factors in the whole test, which also increases the probability of test contamination and manual misoperation

Method used

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  • Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] (1) Using DNAMAN6.0 software, compare the whole genome sequences of TMV, PVY, and CMV virus strains and isolates from various regions included in GenBank, and determine the relatively conserved regions in the virus sequences. Within the selected conserved regions Primers were designed using Oligo 6.0 software, and primer specificity was ensured by BLAST analysis. Since multiplex PCR requires the annealing temperature of each primer and primer pair to be as close as possible, and the size of the amplicon should be as similar as possible under the condition that agarose gel electrophoresis can be clearly distinguished, so a pair of TMV, PVY, and CMV were finally selected. Primers were artificially synthesized and dissolved in RNase-free water to a working concentration of 10 μM.

[0028] TMV: Upstream primer (TF1) 5'-TGTGTGCAAAACTTACTTCCC-3';

[0029] Downstream primer (TR1) 5'-AGGACAAAACATTTGCGTATG-3';

[0030]PVY: upstream primer (PF1) 5'-ACTGTGATGAATGGGCTTATG-3';

...

Embodiment 2

[0038] (1) Using DNAMAN6.0 software, compare the whole genome sequences of TMV, PVY, and CMV virus strains and isolates from various regions included in GenBank, and determine the relatively conserved regions in the virus sequences. Within the selected conserved regions Primers were designed using Oligo 6.0 software, and primer specificity was ensured by BLAST analysis. Since multiplex PCR requires the annealing temperature of each primer and primer pair to be as close as possible, and the size of the amplicon should be as similar as possible under the condition that agarose gel electrophoresis can be clearly distinguished, so a pair of TMV, PVY, and CMV were finally selected. Primers (primers are the same as in Example 1), artificially synthesized and dissolved in RNase-free water to a working concentration of 10 μM.

[0039] (2) Use the TMV, PVY, and CMV virus pathogens preserved in our laboratory to inoculate tobacco at the 6-8 leaf stage (not from the same plant) separatel...

Embodiment 3

[0043] (1) Using DNAMAN6.0 software, compare the whole genome sequences of TMV, PVY, and CMV virus strains and isolates from various regions included in GenBank, and determine the relatively conserved regions in the virus sequences. Within the selected conserved regions Primers were designed using Oligo 6.0 software, and primer specificity was ensured by BLAST analysis. Since multiplex PCR requires the annealing temperature of each primer and primer pair to be as close as possible, and the size of the amplicon should be as similar as possible under the condition that agarose gel electrophoresis can be clearly distinguished, so a pair of TMV, PVY, and CMV were finally selected. Primers (primers are the same as in Example 1), artificially synthesized and dissolved in RNase-free water to a working concentration of 10 μM.

[0044] (2) Use the TMV, PVY, and CMV virus pathogens preserved in our laboratory to inoculate tobacco at the 8-10 leaf stage (tobacco of the same plant) succes...

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Abstract

The method for simultaneously detecting three viruses on tobacco by triple one-step RT-PCR is characterized in that: it is a method for simultaneously detecting tobacco common mosaic virus, potato virus Y and cucumber mosaic virus, and the method includes screening and comparison of conserved virus genomes Region, manual screening and synthesis of compound RT-PCR specific primers suitable for three viral diseases; analysis of TMV, PVY, CMV infected tobacco leaf samples; integration of enzymes and buffers required for reverse transcription and PCR reactions, through optimization of reaction conditions, To establish a method based on one-step RT-PCR combined detection of TMV, PVY and CMV infection in tobacco leaves. The triple one-step RT-PCR reaction adopted in the present invention has the advantages of simple and convenient operation, time-saving and labor-saving, reliable results, and low cost, and can effectively reduce the possibility of cross-contamination of samples during the operation process. , PVY, CMV monitoring and monitoring has a wide range of application prospects.

Description

technical field [0001] The invention belongs to the technical field of plant virus disease detection and provides a triple one-step RT-PCR method for simultaneous detection of tobacco common mosaic virus disease ( Tobacco mosaic virus , TMV), tobacco potato virus Y ( Potato Y virus , PVY) and tobacco cucumber mosaic virus ( Cucumber mosaic virus , CMV), specifically relates to the synthesis of three tobacco virus-specific nucleotide primer sequences and the effective establishment of the detection method. Background technique [0002] Tobacco common mosaic virus ( Tobacco mosaic virus , TMV), potato virus Y ( Potato virus Y , PVY), cucumber mosaic virus ( Cucumber mosaic virus , CMV) has a wide range of hosts and mainly harms various commercial and horticultural crops such as tobacco, tomato, cucumber, and potato. Its geographical distribution is extremely wide, and it is a worldwide viral disease. The crop production on all continents of the world has suffered heav...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
Inventor 杨军张俊祺罗朝鹏王燃李锋金立锋宋纪真林福呈翟妞周会娜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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