Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes

An assay was developed in which reverse transcription (RT), nested polymerase chain reaction (PCR) and accumulation of amplicon-specific fluorescence could take place in a single, closed reaction tube. The assay, which was classical swine fever virus RNA-specific, was compared with other methods for detection of this virus, including various RT-PCR configurations, virus isolation and ELISA. The new method was very sensitive, and less prone to giving false positive results compared to nested PCR carried out in separate reaction tubes. Substitution of different fluorescent probes resulted in specific tests far border disease virus and for bovine viral diarrhoea type II (BVD-II), and one that could detect all pestiviruses except for some BVD-II viruses. Crown Copyright (C) 1999 Published by Elsevier Science B.V. All rights reserved.