Proposal of a Validation Method for Automated Nucleic Acid Extraction and RT -qPCR Analysis: an Example with Bluetongue Virus

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 quantitative polymerase chain reac­tion assays (RT-qPCR) are described for the detection of 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 is 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 low with a total coef­ficient of variation of 1-2% for clear positive samples, and below 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 the same diagnostic results (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.