Appeal point
Method for fragmenting double-stranded RNA capable of detecting unknown viral sequence
Summary
Although many RNA viruses have a dsRNA state in their life cycle, healthy plants, animals, and fungal cells rarely contain dsRNA. Therefore, RNA viruses can be analyzed by extracting and purifying only dsRNA and performing sequence analysis. The present invention is a method capable of detecting an unknown virus sequence and efficiently detecting or searching for a virus. It consists of the following steps.
1) The target double-stranded (ds) RNA is randomly fragmented to obtain a dsRNA fragment.
2) The obtained dsRNA fragment is subjected to reverse transcription reaction and subsequent polymerase chain reaction (PCR) to obtain the corresponding DNA fragment.
3) The obtained DNA fragment is subjected to a sequence analysis operation to determine the sequence.
The figure is a mapping result when the present invention is used for the genomic sequencing of a known dsRNA virus (MoCV1-A). Here, a rice blast fungus infected with MoCV1-A, which is an RNA virus consisting of 5 dsRNAs (3554, 3250, 3074, 3043, 2879 nt), was used as a sample. The full-length genomic sequence of MoCV1-A could be reconstructed by connecting the obtained sequence data. In addition, the values of coverage in the terminal regions of each segment genome tended to be higher than those in the central region.
1) The target double-stranded (ds) RNA is randomly fragmented to obtain a dsRNA fragment.
2) The obtained dsRNA fragment is subjected to reverse transcription reaction and subsequent polymerase chain reaction (PCR) to obtain the corresponding DNA fragment.
3) The obtained DNA fragment is subjected to a sequence analysis operation to determine the sequence.
The figure is a mapping result when the present invention is used for the genomic sequencing of a known dsRNA virus (MoCV1-A). Here, a rice blast fungus infected with MoCV1-A, which is an RNA virus consisting of 5 dsRNAs (3554, 3250, 3074, 3043, 2879 nt), was used as a sample. The full-length genomic sequence of MoCV1-A could be reconstructed by connecting the obtained sequence data. In addition, the values of coverage in the terminal regions of each segment genome tended to be higher than those in the central region.
Schematic image
Application
Life sciences and medical care
Relational patent
Item name
:
Method for fragmenting double-stranded RNA and use thereof
Filing date
:
2015-10-13
Inventor(s)
Syunichi Urayama
Takuo Nunoura
Shigeru Deguchi