A TaqMan real-time RT-PCR assay for selective detection of atypical bovine pestiviruses in clinical samples and biological products.

Novel 'atypical' pestiviruses have been detected recently in biological products, e.g. fetal calf serum (FCS) batches, and in cattle infected naturally. Due to genetic and antigenic variation in pestiviruses, the current diagnostic assays have limitations for the detection of atypical bovine pestiviruses described recently. This paper describes a new one-step real-time RT-PCR assay for the specific detection of atypical bovine pestiviruses, including D32/00_'HoBi', Brz buf 9, CH-KaHo/cont, and Th/04_KhonKaen viruses. The assay detects around 200 copies of synthetic viral RNA molecules per reaction. Coefficient variation (CV) values ranged from 0.13 to 2.11% in three tests performed within 5 weeks, showing that this assay is highly reproducible. To evaluate the suitability for specific detection and identification of the atypical bovine pestiviruses, the assay was evaluated on 46 clinical samples, five batches of FCS and one live Theileria annulata vaccine. Five clinical samples and four batches of commercial FCS were positive for atypical pestiviruses. This new assay provides a useful tool for highly sensitive and specific detection of atypical bovine pestiviruses in clinical samples and can be applied in combination with other diagnostic methods to ensure that biological products, including FCS and vaccines, are free from contamination with pestiviruses.