Validation of a reverse transcriptase multiplex PCR test for the serotype determination of US isolates of bluetongue virus

Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants pri marily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemi ology of the virus, but current methods are cumbersome. A single-tube multi plex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previ ously developed for the serotype determination of U.S. BTV isolates, was ev aluated. The determination of serotype was based on the size of the resulta nt amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a cl osely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates origina ting from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of th e isolates were determined by both a virus neutralization (VN) procedure an d the mRT-PCR. Comparison of the mRT-PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and w as shown to be more reliable and specific than the VN assay. Published by E lsevier Science B.V.