Reagent assisting in identifying sowbane mosaic virus (SoMV) and application thereof

A grass mosaic virus and mosaic virus technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of cumbersome ELISA operation steps, expensive electron microscopy equipment, and low detection sensitivity. Achieve the effects of avoiding false positive results, wide application range and prospects, and improving sensitivity

Inactive Publication Date: 2011-04-06
INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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
There is no research report on the detection of SoMV by real-time fluorescent PCR

Method used

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  • Reagent assisting in identifying sowbane mosaic virus (SoMV) and application thereof
  • Reagent assisting in identifying sowbane mosaic virus (SoMV) and application thereof
  • Reagent assisting in identifying sowbane mosaic virus (SoMV) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of reagent

[0035] 1. Design of primers and probes

[0036] According to the conserved region of the polyprotein gene (DQ450973) sequence in the quinoa mosaic virus genome in the US NCBI nucleic acid database, an upstream primer (SoMVF), two downstream primers (SoMVR1 and SoMVR2) and a Taqmam probe were designed (So ​​MVP).

[0037] 2. Composition of reagents

[0038] 1. Composition of Reagent A

[0039] Reagent A consists of SoMVF, SoMVR1 and SoMVP (primers and probes were synthesized by Shanghai Sangon).

[0040] SoMVF (upstream primer): 5'-CCGATGGAACACTTATTCAACAGT-3' (sequence 1 of the sequence listing);

[0041] SoMVR1 (downstream primer): 5'-TGGAGTTGGTGGAGGAAGTACA-3' (sequence 2 of the sequence listing);

[0042] SoMVP (probe): 5'(FAM)-TCGCCGGGTGTTATGAAGTCAGGATC-3'(TAMARA); (The nucleotide sequence is sequence 4 of the sequence listing, the 5' end is marked with the reporter fluorescent dye FAM, and the 3' end is marked with the ...

Embodiment 2

[0050] Embodiment 2, the application of reagent

[0051] Get the grape leaves that infect five kinds of viruses (SoMV, TBRV, SLRSV, TBSV and PRMV) respectively, get the healthy grape leaves as a contrast, use reagent A and reagent B prepared in Example 1 to carry out specificity measurement and sensitivity measurement respectively, and carry out Amplification efficiency comparison of reagent A and reagent B.

[0052] 1. Determination of specificity of reagents

[0053] 1. Total RNA extraction and quality control

[0054] Total RNA was extracted from leaves infected with each virus (5 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.

[0055] 2. Reverse transcription to synthesize cDNA

[0056] The total RNA extracted from the six kinds of leaves in step 1 was reverse-transcribed with...

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Abstract

The invention aims to provide a reagent assisting in identifying a sowbane mosaic virus and application thereof. The reagent provided by the invention comprises a specific primer consisting of deoxyribonucleic acid (DNA) shown as a sequence 1 in a sequence table, DNA shown as a sequence 2 in the sequence table and DNA shown as a sequence 3 in the sequence table. The reagent can also comprise a specific probe of which the nucleotide sequence is shown as a sequence 4 in the sequence table. The sowbane mosaic virus is an important quarantine harmful pest in China. A method for detecting the SoMV by a nested-RT-Realtime polymerase chain reaction (PCR) is established by three nested PCR primers and a TagMan probe. In the method, a nested PCR and real-time fluorescent PCR technology are combined organically; two sets of PCR systems which consist of three primers and a probe are verified mutually, so that the accuracy of a result is enhanced effectively; and detection sensitivity is enhanced effectively by the real-time fluorescent PCR technology. The method is correct, sensitive, simple, convenient and rapid, and the lower detection limit is up to 0.4 fg/mu l of plant total RNA.

Description

technical field [0001] The invention relates to a reagent for assisting identification of quinoa mosaic virus and its application. Background technique [0002] Sowbane mosaic virus (SoMV) is a member of southern bean mosaic virus. The virus is distributed in nearly 30 countries and regions in the world, including Australia, North America, South America, South Africa, Australia, Bulgaria, Canada, the former Czechoslovakia, Italy, Japan, the former Yugoslavia, Austria, Poland, Switzerland, Germany, Belgium, the Netherlands, United Kingdom, Ireland, France, Greece, Portugal, Spain, Romania, Hungary, Albania, Turkey, Morocco. This virus has not been reported in China, and it is one of the important quarantine virus species imported into my country ("List of Plant Quarantine Pests Imported by the People's Republic of China" (2007 latest edition).). There are many natural hosts of quinoa mosaic virus, such as apples, grapes, cherries, carnations, chrysanthemums, etc. These host...

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 粟智平杨益娥缪玉刚方绍庆耿金培王真邵立洪
Owner INSPECTION & QUARANTINE TECH CENT OF YANTAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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