title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription.
abstract: 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.
authors.affiliation: Laboratory of Veterinary Radiology, School of Veterinary Medicine, Rakuno Gakuen University Ebetsu 069-8501, Japan. dendoh@rakuno.ac.jp
authors.forename: Daiji
authors.initials: D
authors.lastname: Endoh
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mizutani
authors.forename: Rikio
authors.initials: R
authors.lastname: Kirisawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Maki
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Saito
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kon
authors.forename: Shigeru
authors.initials: S
authors.lastname: Morikawa
authors.forename: Masanobu
authors.initials: M
authors.lastname: Hayashi
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D016679:Genome, Viral; D009711:Nucleotides / Q000032:analysis; D029101:Parainfluenza Virus 3, Bovine / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D048348:Reverse Transcription; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000302:isolation & purification; D017423:Sequence Analysis, RNA
chemical_list: D018076:DNA, Complementary; D009711:Nucleotides; D012335:RNA, Ribosomal; D012367:RNA, Viral