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Double-stranded RNA fragmentation method and use thereof

A technology of fragmentation and RNA virus, applied in the field of fragmentation and utilization of double-stranded RNA

Active Publication Date: 2018-09-07
JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent Document 1 proposes a cleavage method for removing a phosphate group from the 3' end of a nucleic acid molecule for an enzyme detection method, but not for sequence analysis

Method used

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  • Double-stranded RNA fragmentation method and use thereof
  • Double-stranded RNA fragmentation method and use thereof
  • Double-stranded RNA fragmentation method and use thereof

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Embodiment

[0139] The techniques disclosed below represent one of the aspects of the present invention for the purpose of being supported by experiments, which can be understood by those skilled in the art. The protection scope of the present application shall be interpreted according to the claims, and shall not be limited to the manner described in the item of the embodiment.

[0140] [Materials and methods]

[0141]

[0142] The rice blast fungus S-0412-II 1a infected with Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) was planted in YG liquid medium (0.5% yeast extract, 2% glucose), at 25°C, 60r .p.m was cultured for 2 weeks with back-and-forth shaking. (this bacterium is to operate with the rice blast fungus produced in Vietnam, because it needs permission, so it is carried out under Professor Teraoka of Tokyo University of Agriculture and Technology with permission. In addition, about MoCV1-A, there are related patents or patent applications (such as , EP2679675, US2011-00...

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Abstract

The present invention addresses the problem of providing a method capable of detecting unknown virus sequences and that can efficiently perform virus detection and searches. The method includes a stepfor randomly fragmenting target double-stranded (ds) RNA to obtain dsRNA fragments; a step for providing the obtained dsRNA fragments to a reverse transcription reaction, followed by a polymerase chain reaction (PCR), to obtain corresponding DNA fragments; and a step for providing the obtained DNA fragments to a sequence analysis operation to determine sequences. The reverse transcription reaction is preferably initiated from the 3' terminal of the dsRNA fragments.

Description

technical field [0001] The present invention relates to a method for fragmenting double-stranded RNA and sequence determination using the same. The present invention can be used for the sequence determination of the full-length genome of RNA viruses, or the detection of known or unknown viruses. The present invention is useful in fields such as life sciences and medicine. Background technique [0002] Virus detection and exploration are important for understanding the role of viral ecology and for controlling viral infections. Conventional virus detection methods include a method based on the detection of a specific viral protein using an antigen-antibody reaction, and a method based on the detection of the nucleotide sequence of a specific viral gene. In addition, RNA sequencing (RNA sequencing, RNA-Seq) is a general method for RNA virus metagenomics (metagenomics), and is widely used in the identification or exploration of RNA viruses. This method is a method of compreh...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/686G01N33/53G01N33/569C12N7/00G16B30/10G16B30/20
CPCC12N7/00C12N15/1096C12Q1/70C12Q1/686C12Q1/6806C12Q2521/107C12Q2531/113C12Q2521/501C12Q2525/191C12Q2525/301G16B30/00C12N15/09G01N33/53C12Q1/68G01N33/569G16B30/10G16B30/20C12N7/02C12P19/34C12Q1/6869C40B50/06
Inventor 浦山俊一布浦拓郎出口茂
Owner JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY
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