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RNA probe for specific detection of cucumber green mottle mosaic virus and preparation method of RNA probe

A technology of RNA probe and green mottled flower, applied in the field of RNA probe for specific detection of cucumber green mottle mosaic virus and its preparation

Pending Publication Date: 2017-11-21
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the above method is used for qualitative and quantitative analysis of viral genomic RNA (gRNA) and subgenomic RNA (sgRNA), there are high requirements for the accumulation and integrity of RNA.

Method used

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  • RNA probe for specific detection of cucumber green mottle mosaic virus and preparation method of RNA probe
  • RNA probe for specific detection of cucumber green mottle mosaic virus and preparation method of RNA probe
  • RNA probe for specific detection of cucumber green mottle mosaic virus and preparation method of RNA probe

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Experimental program
Comparison scheme
Effect test

Embodiment

[0028] Main test materials

[0029] The source of CGMMV was collected from Fengcheng City, Liaoning Province; M-MuLV First Strand cDNA Synthesis Kit reverse transcription kit was purchased from Shanghai Shenggong Biotechnology Co., Ltd.; GXL DNA Polymerase, vector pUC119, endonucleases SacⅠ and BamHI were purchased from Takara Bio Inc. (Dalian Bao Biological Engineering Co., Ltd.); Trizonreagent was purchased from Kangwei Century Biotechnology Co., Ltd.; Dig Northern Start Kit, Dig High Primer DNA Labeling and Detection Starter Kit II was purchased from Roche Bioreagent Co., Ltd. in Germany; the nylon membrane was a product of GE in the United States; CDP-Star Reagent was a product of NEB in the United States; the others were all domestic or imported analytical products.

[0030] Primer design

[0031] The full-length CGMMV genome sequence (GenBank ID: AY859497) was obtained from GenBank, and specific primers were designed and synthesized by Shenggong Bioengineering (Shanghai) Co.,...

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Abstract

The invention discloses an RNA probe for specific detection of cucumber green mottle mosaic virus. The RNA probe is specifically combined with a positive-sense strand RNA of the cucumber green mottle mosaic virus, and detection is performed in a Northern hybridization mode, so that the detection specificity and sensitivity are remarkably improved. The designed RNA probe is used for detecting the accumulation and the integrity of a viral genome RNA (gRNA), a subgenome RNA (sgRNA), defective interfering (DI) RNA and other viral RNA fragments. Through gel electrophoresis to membrane hybridization by a Northern hybridization detection system, the advantages that gRNA, sgRNA and the like of viruses are quantitatively detected, and meanwhile, whether nucleic acid is degraded or not can be judged.

Description

Technical field [0001] The invention relates to the technical field of plant virus detection, in particular to an RNA probe for specifically detecting cucumber green mottle mosaic virus and a preparation method thereof. Background technique [0002] my country's watermelon industry has developed rapidly in recent years and has become the country with the largest watermelon cultivation area in the world. Cucumber green mottle mosaic virus (CGMMV) is distributed all over the world. It mainly harms watermelon and cucurbitaceous crops such as watermelon and cucumber. It causes the symptoms of watermelon inverted flesh (blood flesh). Excessive consumption can cause symptoms such as diarrhea and fever. The current main detection methods of CGMMV mainly include enzyme-linked immunosorbent assay (ELISA) and reverse transcription PCR (RT-PCR). However, when the above methods perform qualitative and quantitative analysis of viral genomic RNA (gRNA) and subgenomic RNA (sgRNA), there are hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/70C12R1/94
CPCC12N15/70C12Q1/6811C12Q1/701C12Q2600/166
Inventor 安梦楠夏博夏子豪赵秀香吴元华董蕴琦程璐侯慧慧
Owner SHENYANG AGRI UNIV
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