BlueTYPE - A low density TaqMan-RT-qPCR array for the identification of all 24 classical Bluetongue virus serotypes

Bluetongue virus is a double-stranded RNA virus with 10 genome segments. VP2 is the primary target for neutralising antibodies and defines the serotype. Today, more than 27 serotypes are known, 24 are defined as classical, and new serotypes are under investigation. Beside group-specific BTV-genome detection, additional serotype characterisation is important for disease control and epidemiological investigations. Therefore, a low-density RT-qPCR array representing a panel of group- and serotype-specific assays, was combined with an internal control system. For BTV serotype detection, both published and the newly developed in-house PCR systems were combined. The different primer-probe-mixes were placed in advance into a 96-well plate stored at -20 degrees C until use. At the time of analysis, the only template RNA was added to the prepared primer-probe-mixes and heat denatured at 95 degrees C for 3 min. After cooling, the master mix was added to each well and the PCR could run for around 90 min. The presented low-density TaqMan-RT-qPCR array enables fast and precise characterisation of the BTV serotype in clinical cases. Furthermore, mixed infections can be easily identified. In addition, the newly developed low-density RT-qPCR-array can easily be adapted to novel BTV strain variants or extended for relevant differential diagnosis.