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

An assay was developed in which reverse transcription (RT), nested polymera se chain reaction (PCR) and accumulation of amplicon-specific fluorescence could take place in a single, closed reaction tube. The assay, which was cl assical swine fever virus RNA-specific, was compared with other methods for detection of this virus, including various RT-PCR configurations, virus is olation and ELISA. The new method was very sensitive, and less prone to giv ing false positive results compared to nested PCR carried out in separate r eaction tubes. Substitution of different fluorescent probes resulted in spe cific 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-I I viruses. Crown Copyright (C) 1999 Published by Elsevier Science B.V. All rights reserved.