Isothermal amplification of rabies virus gene.
A sensitive and specific in situ amplification technique is needed to elucidate the dynamics of rabies virus in the body during the long incubation period after infection. To overcome the disadvantage of using the traditional reverse transcription (RT)-PCR in in situ studies, an isothermal nucleic acid sequence-based amplification (NASBA) technique was developed for detection of the rabies virus gene. The NASBA technique involves the use of 4 enzymatic activities to produce multiple RNA copies of the target sequence by means of double-strand cDNA intermediates under an isothermal condition without thermocycling. The amplified cDNA intermediates from the genomic RNA in the rabies virion and the total RNA in the infected cells in NASBA reaction were analyzed by Southern hybridization assays. The specific amplified products of the rabies viral gene with the expected length were detected after 8 hr of incubation in NASBA using both of the RNAs as templates. The NASBA system used in this study was less sensitive than the general RT-PCR technique. This may have been because we employed Southern hybridization for the amplified cDNA intermediates, not many RNA copies, to evaluate the NASBA results. In conclusion, we successfully amplified the rabies viral gene in the NASBA reaction under an isothermal condition. The unique character of this technique would make it particularly valuable for in situ studies not only on rabies virus but also on other RNA viruses.
Sugiyama
Makoto
M
Laboratory of Zoonotic Diseases, Faculty of Agriculture, Gifu University, Yanagido, Gifu, Japan.
Ito
Naoto
N
Minamoto
Nobuyuki
N
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D006224:Cricetinae; D005784:Gene Amplification / Q000235:genetics*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011820:Rabies virus / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D012367:RNA, Viral