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. 2015 Nov:224:77-82.
doi: 10.1016/j.jviromet.2015.08.013. Epub 2015 Aug 20.

Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains

Michele Losurdo  1 , Viviana Mari  1 , Maria Stella Lucente  1 , Maria Loredana Colaianni  2 , Iolanda Padalino  2 , Nicola Cavaliere  2 , Canio Buonavoglia  3 , Nicola Decaro  4
Affiliations

Development of a TaqMan assay for sensitive detection of all pestiviruses infecting cattle, including the emerging HoBi-like strains

Michele Losurdo et al. J Virol Methods. 2015 Nov.

Abstract

A real-time RT-PCR assay based on the TaqMan technology was developed for rapid and sensitive detection of pestiviruses infecting cattle, i.e., bovine viral diarrhea virus (BVDV) 1, BVDV-2, and the emerging HoBi-like pestiviruses. The assay was linear and reproducible, being able to detect as few as 10 copies of viral RNA. By real-time RT-PCR analysis of 986 biological samples collected from cattle herd with clinical signs suggestive of pestivirus infection and from animals recruited in a pestivirus surveillance programme, 165 pestivirus positive samples were detected, including 6 specimens, 2 nasal swabs, and 4 EDTA-blood samples, that tested negative by a gel-based RT-PCR assay targeting the 5'UTR. The developed TaqMan assay represents a new reliable and effective tool for rapid and sensitive diagnosis of infections caused by all pestiviruses circulating in cattle, thus being useful for extensive surveillance programs in geographic areas where HoBi-like pestiviruses are co-circulating with BVDV-1 and BVDV-2.

Keywords: Cattle; HoBi-like viruses; Pestivirus; Real-time RT-PCR.

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Figures

Fig. 2
Fig. 2
Standard curves obtained for the panpestivirus real-time RT-PCR assay. The dilutions of standard RNA are indicated on the x-axis, whereas the corresponding cycle threshold (CT) values are presented on the y-axis. Each dot represents the result of duplicate amplifications of each dilution. The coefficient of determination (R2) and the slope value (s) of the regression curve were calculated and are indicated.
Fig. 3
Fig. 3
Comparison between panpestivirus gel-based and real-time RT-PCR assays carried out on bovine fetal tissues (A), nasal swabs (B), feces (C), and EDTA–blood samples (D). Numbers indicate the samples positive (+) or negative (−) for pestiviruses. Results according to both techniques are shown in bold.
Fig. 1
Fig. 1
Nucleotide alignment of reference bovine pestiviruses showing the binding region of oligonucleotides used in the panpestivirus real-time RT-PCR.
See this image and copyright information in PMC

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