Emergence of Bluetongue Virus Serotypes in Europe. Part I. Description and Validation of Four Real-Time RT-PCR Assays

Abstract

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.