Real-time reverse transcriptase PCR for the detection of bluetongue virus
In recent years, real-time reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods for the diagnosis of infectious pathogens. The combined properties of high sensitivity, specificity, and speed, along with a low contamination risk, have made real-time PCR technology a highly attractive alternative to more conventional diagnostic methods. Numerous robust rRT-PCR systems have been developed and validated for important epizootic diseases of livestock, and in this chapter we describe an rRT-PCR protocol for the detection of bluetongue virus. The assay uses oligonucleotide primers to specifically amplify target regions of the viral genome and a dual-labeled fluorogenic (TaqMan()) probe which allows for the assay to be performed in a closed-tube format, thus minimizing the potential for cross-contamination.