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Reagent for assisting identification of arabis mosaic virus and application thereof

An auxiliary identification and leaf virus technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of complicated ELISA technology operation steps, expensive electron microscope technology equipment, prone to false positives, etc. Broad application range and prospects, avoid false positive results, reduce the effect of false positive results

Inactive Publication Date: 2013-06-26
粟智平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Electron microscopy equipment is expensive and has low detection sensitivity; ELISA technology has cumbersome operation steps, long detection cycle, low sensitivity, and prone to false positives
Compared with ELISA, PCR gel electrophoresis technology has improved in many aspects, but it is easy to cause pollution
There is no research institute using semi-nested RT-Realtime PCR to detect Armv

Method used

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  • Reagent for assisting identification of arabis mosaic virus and application thereof
  • Reagent for assisting identification of arabis mosaic virus and application thereof
  • Reagent for assisting identification of arabis mosaic virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of reagent

[0036] 1. Design of primers and probes

[0037] Three primers (two upstream primers ArMVF1, ArMVF2 and one downstream primer ArMVR) and one Taqmam probe (ArMVP) were designed according to the conserved region of the polyprotein gene sequence in the A. thaliana mosaic virus genome in the American NCBI nucleic acid database. .

[0038] 2. Composition of reagents

[0039] 1. Composition of Reagent A

[0040] Reagent A consists of ArMVF1, ArMVR and ArMVP (primers and probes were synthesized by Shanghai Sangon).

[0041] ArMVF1 (upstream primer): 5'-TGCTGAGTTTGAGGCAGCAA-3' (sequence 1 of the sequence listing);

[0042] ArMVR (downstream primer): 5'-CAGTGGTGGGATGGTCAGAAA-3' (sequence 2 of the sequence listing);

[0043] ArMVP (probe): 5'(FAM)-CGACTTTGCCAGCCTAGATGCAG-(TAMRA)3'; (The nucleotide sequence is sequence 4 of the sequence listing, the 5' end is labeled with the reporter fluorescent dye FAM, and the 3' end is labeled wit...

Embodiment 2

[0051] Embodiment 2, the application of reagent

[0052] Infected with four viruses (ArMV, PVX, TBRV and PVY) N ) of potato leaves, healthy potato leaves were taken as a control, and reagent A and reagent B prepared in Example 1 were used for specificity determination and sensitivity determination respectively, and the amplification efficiencies of reagent A and reagent B were compared.

[0053] 1. Determination of specificity of reagents

[0054] 1. Total RNA extraction and quality control

[0055] Total RNA was extracted from leaves infected with each virus (4 species) and healthy leaves (CK1) with a plant total RNA extraction kit. Use the nucleic acid protein analyzer BioPhotometer to measure its OD value, get its concentration and purity value, and use this to control the quality of nucleic acid.

[0056] 2. Reverse transcription to synthesize cDNA

[0057] The total RNA extracted from the five kinds of leaves in step 1 was reverse-transcribed with a reverse transcript...

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Abstract

The invention aims at providing a reagent for assisting the identification of arabis mosaic virus and an application thereof. The reagent provided by the invention comprises a specific primer consisting of DNA (Deoxyribonucleic Acid) shown in a sequence 1 in a sequence table, DNA shown in a sequence 2 in the sequence table, and DNA shown in a sequence 3 in the sequence table. The reagent also comprises a specific probe shown in a sequence 4 in a nucleotide sequence table. The arabis mosaic virus is an important quarantine pest in China, and a method for detecting ArMV by semi-nested-RT (Reverse Transcriptase)-Realtime PCR (Polymerase Chain Reaction) is established by utilizing three nested type PCR primers and a TaqMan Probe. According to the method, the nested PCR technology and the real-time fluorescence PCR technology are organically combined; two PCR systems formed by the three primers and the probe are verified with each other, so that the result accuracy is effectively improved; the detection flexibility is effectively improved by the real-time fluorescence PCR technology; and the accuracy, the flexibility, the simplicity, the convenience and the rapidness are realized, and the minimum detection limit can reach 0.5fg / mul plant total RNA (Ribonucleic Acid).

Description

technical field [0001] The invention relates to a reagent for assisting identification of Arabidopsis mosaic virus and its application. Background technique [0002] Arabis mosaic virus (ArMV) is a member of the genus Nepovirus of the family Comoviridae. Its geographical distribution is extremely wide, occurring in more than 30 countries on five continents, and it is a worldwide disease. There is no report on the harm of this virus in our country, and it is an important quarantine plant virus in my country. [0003] The virus has a wide host range and can infect about 174 genera and 215 species of plants. It mainly harms economic crops such as cucumbers, lettuce, potatoes, carrots, tulips, tobacco, cherries, grapes, strawberries, spinach, roses, soybeans, and kidney beans. [0004] ArMV can be transmitted over long distances through vegetative propagation materials such as seeds, scales, tubers and seedlings. In the field juice, grafting, etc. can spread the virus. Seeds...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N21/64
Inventor 粟智平耿金培刘爱华张苗王建力杨益娥李金庆
Owner 粟智平
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