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Nucleic Acids Research 2005 33(6):e65; doi:10.1093/nar/gni064
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Published online 7 April 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

Daiji Endoh*, Tetsuya Mizutani1, Rikio Kirisawa2, Yoshiyuki Maki3, Hidetoshi Saito3, Yasuhiro Kon4, Shigeru Morikawa1 and Masanobu Hayashi

Laboratory of Veterinary Radiology, School of Veterinary Medicine, Rakuno Gakuen University Ebetsu 069-8501, Japan 1Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases Musashimurayama 208-0011, Japan 2Laboratory of Veterinary Microbiology, School of Veterinary Medicine, Rakuno Gakuen University Ebetsu 069-8501, Japan 3Genosys Division, Sigma-Aldrich Japan Ishikari 061-3241, Japan 4Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University Sapporo 060-0818, Japan

*To whom correspondence should be addressed. Tel: +81 11 388 4847; Fax: +81 11 387 5890; Email: dendoh{at}rakuno.ac.jp

Received December 10, 2004. Revised February 26, 2005. Accepted March 18, 2005.

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)+ RNAs but not for poly(A)– RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.


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