A single-tube RT-PCR method for the detection of Borna disease viral genomic RNA.

Abstract

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.

• Authors: T Mizutani, M Ogino, Y Nishino, T Kimura, H Kariwa, K Tsujimura, H Inagaki, I Takashima
• Journal: The Japanese journal of veterinary research (Jpn J Vet Res) Vol. 46 Issue 2-3 Pg. 73-81 (Nov 1998) ISSN: 0047-1917 [Print] Japan
• PMID: 10093417 (Publication Type: Journal Article)

Chemical References

• DNA Primers
• RNA, Viral

Topics

• Animals
• Base Sequence
• Borna Disease (diagnosis)
• Borna disease virus (isolation & purification)
• Brain (virology)
• Cell Line
• DNA Primers
• Dogs
• Kidney
• Molecular Sequence Data
• RNA, Viral (analysis)
• Rats
• Reverse Transcriptase Polymerase Chain Reaction (methods)