Emergence des sérotypes de fièvre catarrhale ovine en Europe. Partie I - Description et validation de quatre tests diagnostiques par RT-PCR en temps réel

Résumé

The control of bluetongue virus (BTV) in Central Western Europe is greatly complicated by the coexistence of several BTV serotypes. Rapid, sensitive and specific assays are there­fore needed to identify correctly the currently circulating BTV serotypes in field samples. In the present study, four serotype-specific real-time reverse transcription - polymerase chain reac­tion (RT-qPCR) assays are described for the detection of BTV serotype 1 (BTV-1), 6, 8 and 11. The analytical sensitivity of BTV-1/S2, BTV-6/S2, BTV-8/S2 and BTV-11/S2 serotype-specific RT-qPCR assays was comparable to the earlier described sero­group-specific pan-BTV/S5 RT-qPCR assay. In silico and in vitro analyses indicated that none of the assays cross-reacted with viruses which were symptomatically or genetically related to BTV, and only detected the intended BTV serotypes. All assays exhibited a linear range of at least 0.05–3.80 log₁₀ TCID₅₀ mL) and a PCR-efficiency approaching the ideal amplification factor of two per PCR cycle. Both intra- and inter-run variations were found to be low with a total coefficient of variation of 1–2% for clear positive samples and < 10% for very weak positive samples. Finally, the performance of the described assays was compared with commercially available kits for the detection of BTV-1, 6 and 8. Three in-house assays gave exactly the same diagnostic result (positive/negative) as the commercial assays and can thus be used interchangeably. Together with the earlier described serogroup-specific pan-BTV/S5, the serotype-specific RT-qPCR assays form a flexible and properly validated set of tools to detect and differentiate the BTV serotypes currently circulating in Central Western Europe.