In the last decade, real time PCR has been increasingly adopted for
bluetongue diagnosis with both broadly reactive and serotype-specific
assays widely used. The use of these assays and nucleic acid sequencing
technologies have enhanced bluetongue virus detection, resulting in the
identification of a number of new serotypes. As a result, more than 30
different serotypes are proposed. Rapid identification of the virus
serotype is essential for matching of antigens used in vaccines and to
undertake surveillance and epidemiological studies to assist risk
management. However, it is not uncommon for multiple serotypes to
circulate in a region either concurrently or in successive years. It is
therefore necessary to have a large suite of assays available to ensure
that the full spectrum of viruses is detected. Nevertheless, covering a
large range of virus serotypes is demanding from both a time and
resource perspective. To overcome these challenges, real time PCR assays
were optimised to match local virus strains and then combined in a panel
of quadriplex assays, resulting in 3 assays to detect 12 serotypes
directly from blood samples from cattle and sheep. These multiplex
assays have been used extensively for bluetongue surveillance in both
sentinel animals and opportunistically collected samples. A protocol to
adapt these assays to capture variations in local strains of bluetongue
virus and to expand the panel is described. Collectively these assays
provide powerful tools for surveillance and the rapid identification of
bluetongue virus serotypes directly from animal blood samples.