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

A RT-PCR and virus technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of increasing test pollution, manual misoperation, and many influencing factors, and reduce sample crossover Pollution, reduction of influencing factors, reliable results

Active Publication Date: 2011-11-16
ZHENGZHOU TOBACCO RES INST OF CNTC
View PDF5 Cites 10 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction)
  • 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

[0030] (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.

[0031] TMV: Upstream primer (TF1) 5'-TGTGTGCAAAACTTACTTCCC-3';

[0032]Downstream primer (TR1) 5'-AGGACAAAACATTTGCGTATG-3';

[0033] PVY: upstream primer (PF1) 5'-ACTGTGATGAATGGGCTTATG-3';

...

Embodiment 2

[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 6-8 leaf stage (not from the same plant) separatel...

Embodiment 3

[0049] (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.

[0050] (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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction). The method is characterized by being used for synchronously detecting TMV (Tobacco Mosaic Virus), PVY (Potato Virus Y) and CMV (Cucumber Mosaic Virus), the method comprises the following steps of screening and comparing conservation areas in a virus gene group, artificially screening and synthesizing specificity primers suitable for composite RT-PCR of three kinds of virus diseases; analyzing to obtain TMV, PVY and CMV infection tobacco leaf samples; and integrating enzymes and a buffer solution required by a reverse transcription and a PCR (Polymerase Chain Reaction), and establishing a method for compositely detecting the TMV, PCY and CMV infection of tobacco leaves based on one-step method RT-PCR through optimization of reaction conditions. The triple one-step method RT-PCR utilized by the invention has the advantages of simple and convenient operation, time and labor saving, reliable result and low cost; simultaneously, the probability of cross-contamination of samples in an operation process canbe effectively reduced, and the monitoring on the TMV, PVY and CMV in tobacco agricultural production has wide application prospect.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap