Method for compositely detecting tobacco common mosaic virus and potato virus Y in one step and primers thereof

A technology for compound detection and mosaic virus, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc. It can solve the problems of high requirements for virus sample materials, limited experimental conditions, and limited wide application. , to achieve the effects of reducing influencing factors, ensuring accuracy, and high virus pathogen specificity

Active Publication Date: 2011-08-03
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Abstract
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Problems solved by technology

Routine biological detection is a basic link in the research of plant viruses, mainly including determination of host range, identification of host response, temperature of virus sap, dilution limit, in vitro survival period, determination of in vitro traits, and determination of transmission routes, etc., but it is time-consuming , Fei Li (Liang Xunsheng, Xie Lianhui. Plant Virology [M]. Beijing: China Agricultural Press, 1994)
The newly developed detection

Method used

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  • Method for compositely detecting tobacco common mosaic virus and potato virus Y in one step and primers thereof
  • Method for compositely detecting tobacco common mosaic virus and potato virus Y in one step and primers thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Use DNAMAN6.0 to compare the whole genome sequences of TMV and PVY virus strains and isolates from different regions included in GenBank, and determine the relatively conserved regions in the virus sequences. Primers were designed using Primer Premier software in the selected conserved regions, and analyzed with BLAST tool (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to ensure primer specificity. Finally, two pairs of primers were selected. PVY: Upstream primer (PF) 5'-GGATCAATGCATCCTGTCATAC-3'; Downstream primer (PR) 5'-GCYTTGTCAGACCAATCTTTCC-3'. TMV: Upstream primer (TF) 5'-CGGAGATCTCGACAGTCATGTG-3'; Downstream primer (TR) 5'-TACAGGCGTAAACATCGACGG-3'. The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and dissolved in RNase-free water according to the synthesis instructions to a working concentration of 20 μm;

[0022] (2) Use Agdia’s TMV and PVY diagnostic kits (PathoScreen TMV, PathoScreen PVY) detection and analysis of virus tobacco leaf...

Embodiment 2

[0028] (1) Artificially synthesize two pairs of virus-specific primers. PVY: upstream primer (PF) 5'-GGATCAATGCATCCTGTCATAC-3'; downstream primer (PR) 5'-GCYTTGTCAGACCAATCTTTCC-3'. TMV: Upstream primer (TF) 5'-CGGAGATCTCGACAGTCATGTG-3'; Downstream primer (TR) 5'-TACAGGCGTAAACATCGACGG-3'. Dissolve in RNase-free water to a working concentration of 20 μm according to the synthesis instructions;

[0029] (2) Use Agdia’s TMV and PVY diagnostic kits (PathoScreen TMV, PathoScreen PVY) detection and analysis of virus tobacco leaf samples collected in Daejeon Tobacco Area, and the tobacco leaf samples infected with TMV and PVY were found by serological methods;

[0030] (3) Extraction of total RNA from tobacco leaves

[0031] The Ez Spin Column Plant Isolation Kit from Bio Basic Company was used to extract TMV and PVY total RNA respectively.

[0032] (4) Establishment of one-step compound RT-PCR reaction conditions

[0033] The RT-PCR in this experiment uses PrimeScript One Ste...

Embodiment 3

[0035] (1) Artificially synthesize two pairs of virus-specific primers. PVY: upstream primer (PF) 5'-GGATCAATGCATCCTGTCATAC-3'; downstream primer (PR) 5'-GCYTTGTCAGACCAATCTTTCC-3'. TMV: Upstream primer (TF) 5'-CGGAGATCTCGACAGTCATGTG-3'; Downstream primer (TR) 5'-TACAGGCGTAAACATCGACGG-3'. Dissolve in RNase-free water to a working concentration of 20 μm according to the synthesis instructions;

[0036] (2) Use Agdia’s TMV and PVY diagnostic kits (PathoScreen TMV, PathoScreen PVY) detection and analysis of virus tobacco leaf samples collected in Daejeon Tobacco Area, and the tobacco leaf samples infected with TMV and PVY were found by serological methods;

[0037] (3) Extraction of total RNA from tobacco leaves

[0038] The Ez Spin Column Plant Isolation Kit from Bio Basic Company was used to extract the total RNA of TMV and PVY respectively, and mixed in equal amounts as the virus nucleic acid template for one-step compound RT-PCR.

[0039] (4) Establishment of one-step c...

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Abstract

The invention relates to a method for compositely detecting tobacco common mosaic virus and potato virus Y in one step, which is characterized by comprising the following steps of: screening and comparing conservative domains in virus genome, and artificially screening and synthesizing two composite RT(Reverse Transcription)-PCR (Polymerase Chain Reaction) specific primers suitable for viral diseases; analyzing to obtain TMV (Tobacco Mosaic Virus) and PVY (potato virus Y) composite tobacco-infected samples, and integrating enzymes and buffer solution required for reverse transcription and PCR; and then establishing the method for compositely detecting the TMV and the PVY in one step by reaction condition optimization. The method has the advantages that 1) the pathogens of the TMV and the PVY are more convenient to detect, test pollution and influencing factors in the reaction are reduced, and the detection time is greatly shortened; 2) the provided TMY and PVY specific primers have high viral pathogen specificity and can effectively and independently or compositely identify the TMV and the PVY, and ensure the accuracy of the detection result; and 3) when in use, the method saves time and labor, needs low requirement on sample materials, and has less limitation on experimental conditions.

Description

technical field [0001] The invention belongs to the field of plant disease detection and prevention, and is a one-step compound detection method for Tobacco mosaic virus (TMV) and tobacco potato Y virus (Potato Y virus, PVY). Synthesis of specific nucleotide primer sequences and effective establishment of the detection method. Background technique [0002] Tobacco virus disease is the main disease in tobacco agricultural production, and the occurrence of damage will cause a great loss of tobacco yield and lead to a serious decline in the quality of tobacco leaves. Viral diseases are common and serious hazards in tobacco. According to statistics, in 2008, the loss of tobacco leaf output value caused by viral diseases in my country accounted for 23.4% of the total loss of pests and diseases. Viral diseases have become the most serious tobacco diseases (Liu Guoshun, Liu Jianli. China Tobacco Production Practical Technical Guide [M]. Beijing: Agricultural Press, 2007). Tobacco ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12N15/11C12Q1/68
Inventor 杨军张俊祺罗朝鹏宋纪真金立锋李锋翟妞许亚龙林福呈
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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