Bluetongue virus diagnosis of clinical cases by a duplex reverse transcription-PCR: a comparison with conventional methods

A duplex reverse transcription polymerase chain reaction (RT-VCR) assay for the detection of bluetongue virus (BTV) in clinical samples was developed. This assay, which detects the highly conserved S10 region of BTV, was asse ssed for sensitivity and application as a rapid and dependable diagnostic t ool by comparison with standard assays of virus detection, such as virus is olation in embryonated chicken eggs and cell culture. Simultaneous detectio n of BTV and host beta -actin RNAs minimizes the possibility of false negat ive results. The sensitivity of the assay was found to be equal to five cel l culture infectious dose (CCID50) units and its specificity was confirmed as no RT-PCR product was detected with RNAs from two closely related orbivi ruses, i.e. epizootic haemorrhagic disease virus (serotypes 1, 2 and 318) a nd African horse sickness virus, serotype 9, or RNAs from uninfected BHK-21 cells and blood samples from uninfected sheep or goats. In this study, 36 blood samples from naturally infected mixed flocks of sheep and goats were examined. Seventeen animals were identified as BTV-positive by RT-VCR, wher eas only 13 were found positive by virus isolation in embryonated chicken e ggs and nine by cell culture assays. These results indicate that the duplex RT-PCR could be a useful technique for monitoring BTV infection in the fie ld. (C) 2001 Elsevier Science B.V. All rights reserved.