Detection and quantitation of bluetongue virus serotypes by a TaqMan probe-based real-time RT-PCR and differentiation from epizootic hemorrhagic disease virus

Abstract

Twenty-four serotypes of bluetongue virus (BTV) have been recognized world wide. Reliable and quantitative assays of virus universal detection are essential for fighting against BT. A real-time reverse transcription-polymerase chain reaction (RT-PCR) with a TaqMan fluorescence probe has been developed for detection of the NS1 gene of different BTV serotypes. In BHK-21 cells, in the assay detected BTV1-22 specifically, and had no cross-reactivity with the closely related epizootic hemorrhagic disease virus (EHDV) serotypes 1-5. The limit of sensitivity of the assay was 0.1 TCID(50)/ml for BTV-1 and 10(2) copies for the control R121/pGEM. Accurate quantitation can be achieved with samples containing between 10(2) and 10(6) copies. The coefficient of variation (CV) of intra-assay and inter-assay ranged from 2.17% to 5.60%. The developed real-time RT-PCR assay showed good coincident rate (99.2%) with duplex RT-PCR in 122 whole blood clinical samples from sheep. Therefore, the real-time RT-PCR can be a reliable method for detection of various serotypes of BTV. Copyright 2010 Elsevier B.V. All rights reserved.