Real-time polymerase chain reaction to detect bluetongue virus in blood samples

The authors describe a real-time reverse transcriptase polymerase chain reaction (RT-PCR) to detect bluetongue viruses (BTV) in blood samples. The primers and Taqman probes used were specific for a conserved region of BTV RNA segment 5, which encodes non-structural protein NS1. The method was able to detect strains of BTV serotypes 2, 4, 9 and 16 isolated in Italy, and their respective vaccine strains. The limit of detection was 5.0x10-(3) TCID(50) per ml of sample. The assay did not amplify RNA from other Orbiviruses, including epizootic haemorrhagic disease virus (EHDV) or African horse sickness virus (AHSV), or from viruses in the Reoviridae family or those that cause a similar clinical picture to that of BTV. Its accuracy was evaluated on 104 blood samples in ethylenediamine tetra-acetic acid (EDTA) and the results were compared with those obtained with the conventional RT-PCR used in routine diagnosis. Both tests gave negative results on 40 blood samples from bluetongue-free farms (confidence interval: 95%, 92.5-100%). Real-time PCR detected BTV RNA in 64 sentinel cows that had recently seroconverted to serotypes 2 and 16 (confidence interval: 95%, 95.5-100%), whereas conventional RT-PCR detected only 47 of these (confidence interval: 95%, 61.5-82.7%) (P<0.05). The method is rapid, thereby reducing execution times, and does not require any post-amplification manipulation, thus avoiding the inherent risk of contamination of amplified products.