Simultaneous detection of bovine arboviruses using single-tube multiplex reverse transcription-polymerase chain reaction

Single-tube multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay was developed to detect and identify arboviruses in infected cell-culture fluids and field specimens. The technique was equally sensitive for detecting five different viruses in cell cultures, namely the Chuzan. Ibaraki, and Bluetongue viruses belonging to Orbivirus, and the Akabane virus and Peaton virus belonging to Orthobunyovirus, and was less sensitive than former viruses for detecting Aino virus belonging to Orthobunyavirus. The mRT-PCR reliably detected 0.6-10(3.1) median tissue culture infective doses. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. The technique was as sensitive as virus isolation in detecting single infected plasma in five plasmas from sentinel cattle and in detecting two infectious homogenates in eight homogenates of Culicoides biting midges. The mRT-PCR may be a sensitive and rapid assay for surveillance of bovine arboviruses in field specimens. (C) 2004 Elsevier B.V. All rights reserved.