Reverse transcription-nested polymerase chain reaction for detecting p40 RNA of Borna disease virus, without risk of plasmid contamination.

Several methods for the detection of Borna disease virus (BDV) RNA have been reported, one being the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method. However, due to the possibility of contamination of the cloned DNA in a reaction tube, false-positive results might be obtained by RT-nested PCR. To detect only BDV RNA without anxiety of contamination, we developed an RT-nested PCR system using "mRNA selective PCR kit". Using this system, cDNA of BDV p40 in the plasmid (up to 5 x 10(7) molecules) was not amplified. BDV specific sequence was amplified from total RNA (more than 50 pg) of MDCK/BDV cells, which were persistently infected with BDV. These results indicate that this mRNA selective RT-nested PCR system can specifically amplify target RNA as distinguished from plasmid contaminated.