A single-tube RT-PCR method for the detection of Borna disease viral genomic RNA.
For detecting Borna disease virus (BDV) genomic stranded RNA, single-tube reverse transcription-polymerase chain reaction (St RT-PCR) was developed to equal the sensitivity of RT-nested PCR but with reduced risk of contamination. BDV-genomic stranded RNA was synthesized in vitro using plasmid cDNA of BDV p24 region as a template and RNA was also extracted from BDV-persistently infected MDCK (MDCK/BDV) cells. Both RNAs were amplified by St RT-PCR in which a single round of RT and a single round of PCR were performed in the same tube. Ten copies of synthesized RNA could be amplified by St RT-PCR, indicating that St RT-PCR method is as sensitive as the ordinary RT-nested PCR method. Furthermore, this method was applied to quantify the exact copy number of genomic RNA in MDCK/BDV cells. Signals were obtained from the samples containing more than 1 pg total cellular RNA. From the results, approximately 100 copies of BDV genomic RNA exist in one MDCK/BDV cell. BDV genomic RNA from the in vivo RNA samples using St RT-PCR, indicating this method is applicable for the epidemiological study of BDV without contamination.
Mizutani
T
T
Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Ogino
M
M
Nishino
Y
Y
Kimura
T
T
Kariwa
H
H
Tsujimura
K
K
Inagaki
H
H
Takashima
I
I
D000818:Animals; D001483:Base Sequence; D001890:Borna Disease / Q000175:diagnosis*; D001891:Borna disease virus / Q000302:isolation & purification*; D001921:Brain / Q000821:virology; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D007668:Kidney; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000032:analysis*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D012367:RNA, Viral