Recombinant plant dsRNA-binding protein as an effective tool for the isolation of viral replicative form dsRNA and universal detection of RNA viruses
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The isolation of viral replicative form (RF) double-stranded RNA (dsRNA) is a classic technique for plant virus detection when the virus species cannot be predicted from disease symptoms. However, the method has not been very widely used, most likely because dsRNA isolation using CF-11 cellulose is laborious and time-consuming. Here we report an alternative tool, a recombinant plant dsRNA-binding protein, to isolate dsRNA. This tool enables us to isolate viral RF dsRNA in an hour from either extracted nucleic acids or crude detergent extracts. Combining this technique with sequence-non-specific reverse transcription, PCR amplification, cloning, and sequencing, a variety of viruses were efficiently detected using a single set of reagents and procedures.
KeywordsDetection dsRNA dsRNA-binding protein Replicative form RNA virus
We thank Tetsuya Mizutani for advice on the experiments, David Baulcombe for PVX and TRV vectors, Yoshikatsu Genda for PVY, Yasuya Iwadate for TSWV-infected material and Kazue Obara for technical assistance. This study was supported by the Iwate Prefectural Government.
- Chaumpluk P, Sasaki Y, Nakajima N, Nagano H, Nakamura I, Suzuki K, Mise K, Inouye N, Okuno T, Furusawa I (1996) Six new subgroup I members of Japanese cucumber mosaic virus as determined by nucleotide sequence analysis on RNA3’s cDNAs. Ann Phytopathol Soc Jpn 62:40–44Google Scholar
- Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA (eds) (2005) Virus taxonomy: VIIIth Report of the International Committee on Taxonomy of Viruses. Academic Press, New YorkGoogle Scholar
- Kihara Y, Satho T, Eshita Y, Sakai K, Kotaki A, Takasaki T, Rongsriyam Y, Komalamisra N, Srisawat R, Lapcharoen P, Sumroiphon S, Iwanaga S, Ushijima H, Endoh D, Miyata T, Sakata A, Kashige N, Miake F, Fukushi S, Saijo M, Kurane I, Morikawa S, Mizutani T (2007) Rapid determination of viral RNA sequences in mosquitoes collected in the field. J Virol Methods 146:372–374PubMedCrossRefGoogle Scholar