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Preparation method and detection of hybridization probe and gene chip for PPV (plum pox virus) major strains

A gene chip and plum pox virus technology, which is applied in the field of preparation and detection of hybridization probes and gene chips for main strains of plum pox virus, can solve the problems of detection and identification of different strains of plum pox virus that have not been reported.

Active Publication Date: 2016-03-30
陈定虎
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, gene chip technology has been widely used in many aspects, but there are no reports on the detection and identification of the main different strains of plum pox virus.

Method used

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  • Preparation method and detection of hybridization probe and gene chip for PPV (plum pox virus) major strains
  • Preparation method and detection of hybridization probe and gene chip for PPV (plum pox virus) major strains
  • Preparation method and detection of hybridization probe and gene chip for PPV (plum pox virus) major strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] A hybridization probe for main strains of plum pox virus, comprising:

[0079] PPV-D: 5'-CAACAAAACCAGTTTCACAGGTGCCAGGACCTCAACTGCAA-3'

[0080] PPV-M: 5'-CGGTAAGACCAGTTCCTCCAATTTCAGGGACCAAACCGCGG-3'

[0081] PPV-EA: 5'-CCACTAGGACAGTGCCTCACACAACAACTACTACACCTCCT-3'

[0082] PPV-Rec: 5'-CGATAAGACCAGTTTCTCCAATTTCAGGGGCCACACCGCAG-3'

[0083] PPV-W: 5'-GCGTGAAGCCAACTACATCAGCAACAATTAACCCAACGTCT-3'

[0084] PPV-C: 5'-ATGTGAGACCGATTGCACCAGTAGTGACAAGTCCATTCTCG-3'.

Embodiment 2

[0086] A method for preparing a gene chip, comprising the following steps:

[0087] A. Preparation of gene chip spotting:

[0088] Take 5ul of purified probe, add 5ul of spotting solution and mix well, the probe concentration is 10μm; add it to the sample spotter, spot the oligonucleotide probe on the aldehyde-based glass slide, and pass through the amino group at the 5' end of the probe carry out covalent immobilization;

[0089] Wherein said probes include:

[0090] PPV-D: 5'-CAACAAAACCAGTTTCACAGGTGCCAGGACCTCAACTGCAA-3'

[0091] PPV-M: 5'-CGGTAAGACCAGTTCCTCCAATTTCAGGGACCAAACCGCGG-3'

[0092]PPV-EA: 5'-CCACTAGGACAGTGCCTCACACAACAACTACTACACCTCCT-3'

[0093] PPV-Rec: 5'-CGATAAGACCAGTTTCTCCAATTTCAGGGGCCACACCGCAG-3'

[0094] PPV-W: 5'-GCGTGAAGCCAACTACATCAGCAACAATTAACCCAACGTCT-3'

[0095] PPV-C: 5'-ATGTGAGACCGATTGCACCAGTAGTGACAAGTCCATTCTCG-3';

[0096] B. PCR amplification and sample labeling:

[0097] B1. Extraction of total RNA from susceptible plants;

[0098] Weigh 0.1...

Embodiment 3

[0117] A method for preparing a gene chip, comprising the following steps:

[0118] A. Preparation of gene chip spotting:

[0119] Take 5ul of purified probe, add 5ul of spotting solution and mix well, the probe concentration is 10μm; add it to the sample spotter, spot the oligonucleotide probe on the aldehyde-based glass slide, and pass through the amino group at the 5' end of the probe carry out covalent immobilization;

[0120] Wherein said probes include:

[0121] PPV-D: 5'-CAACAAAACCAGTTTCACAGGTGCCAGGACCTCAACTGCAA-3'

[0122] PPV-M: 5'-CGGTAAGACCAGTTCCTCCAATTTCAGGGACCAAACCGCGG-3'

[0123] PPV-EA: 5'-CCACTAGGACAGTGCCTCACACAACAACTACTACACCTCCT-3'

[0124] PPV-Rec: 5'-CGATAAGACCAGTTTCTCCAATTTCAGGGGCCACACCGCAG-3'

[0125] PPV-W: 5'-GCGTGAAGCCAACTACATCAGCAACAATTAACCCAACGTCT-3'

[0126] PPV-C: 5'-ATGTGAGACCGATTGCACCAGTAGTGACAAGTCCATTCTCG-3';

[0127] The specific method of spotting: use a spotting machine to spot samples in an array arrangement, the center distance betwee...

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Abstract

The invention discloses a preparation method of a hybridization probe and a gene chip for PPV (plum pox virus) major strains. The probe comprises PPV-D, PPV-M, PPV-EA, PPV-Rec, PPV-W and PPV-C. The preparation method of a gene chip is characterized by comprising the following steps: gene chip spotting, PCR amplification and sample marking, chip pretreatment, chip hybridization, chip cleaning, chip scanning analysis and data treatment and result judgment. The invention aims at overcoming the shortcomings of the prior art and providing a hybridization probe for detecting the PPV major strains; and the invention also relates to a preparation method of a gene chip comprising the probe.

Description

technical field [0001] The present invention relates to a hybridization probe for the main strains of plum pox virus, the invention also relates to a method for preparing a gene chip comprising the probe, and the invention also relates to a method for detecting plum pox virus by using the above gene chip , belonging to the field of biotechnology. Background technique [0002] Plumpoxvirus, PPV is an entry plant quarantine pest promulgated by my country in 2007. It is one of the most dangerous viruses for stone fruit trees. It belongs to the genus Potyvirus in the family Potyviridae, and its natural hosts are mainly It is a woody plant of the genus Prunus. After infecting drupe fruit trees such as plums, apricots, peaches, and cherries, the leaves and fruits are seriously damaged, and a large number of immature fruits fall off, resulting in a decline in fruit quality and yield. The long-distance spread of the virus mainly depends on the transfer of plant propagation materials...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/70C12Q1/68C12R1/93
Inventor 陈定虎陈祖华刘恭源王振华周敏熊仁广刘晓媚张静冯雪雅管维吴颖儿
Owner 陈定虎
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