Summary
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
5.0×10-3 TCID50 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.
Keywords
Bluetongue
virus, Diagnosis, Polymerase chain reaction, Real-time reverse transcriptase
polymerase chain reaction. |