Nested and multiplex polymerase chain reactions for the identification of bluetongue virus infection in the biting midge, Culicoides variipennis.

Two polymerase chain reaction tests for the detection of bluetongue viral (BLU) RNA in the principal North American insect vector, Culicoides variipennis, were developed. The BLU serogroup specific test used the highly expressed non-structural protein 1 gene as the target gene and two amplification steps. First a 1228 base pair product was amplified using an outer primer pair, then a second amplification using a nested or internal primer pair produced a 930 base pair product. This nested PCR test was found to be very sensitive detecting an equivalent to 1 plaque-forming unit of BLU viral RNA extracted from infected biting midges. The serotype specific test used a multiplex PCR approach in which five different primer pairs were used simultaneously. Each pair was based on the variable outer capsid protein VP2 gene of the five US serotypes generating specific product which were easily identified by size difference. The sensitivity of the multiplex PCR was less sensitive than the nested-PCR but sufficient for use with field collected samples. These tests provide valuable tools for epidemiologic studies of BLU disease.