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 bee n reported, one being the reverse transcription-nested polymerase chain rea ction (RT-nested PCR) method. However, due to the possibility of contaminat ion of the cloned DNA in a reaction tube, false-positive results might be o btained by RT-nested PCR. To detect only BDV RNA without anxiety of contami nation, 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) molecu les) 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 c an specifically amplify target RNA as distinguished from plasmid contaminat ed.