Development of Real-Time Duplex RT-PCR for Thedetection of Bovine Viral Diarrhea Virus and Bovine Rotavirus in Cattle

Bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV) are important pathogens of cattle. These pathogenselicitsigns that is sometimes difficult to differentiate. A real-time duplex RT-PCR assay would allow the simultaneous detection of the two viruses in the same sample. A real-time duplex RT-PCR assay was developed and optimized. The detection limit of the assay was 5 TCID50 or 100 RNA copies of BVDV and 20 TCID50 or 100 RNA copies for BRV. No cross reactivity with other bovine pathogens was detected. Both standard curves have amplification efficiency (E) of 1.06-0.95 and a correlation coefficient (R2) of 0.9917-0.9988, with the dynamic range of detection between 103 to 108copies per reaction. The sensitivity and specificityof this assay were compared with single real-time RT-PCR and conventional RT-PCR assays to verify its accuracy and sensitivity.The standardized duplex realtime RT-PCR confirmed the presence of viral RNA in 26 of 93 clinical samples. Two clinical samples were shown to be co-infected with both BVDV and BRV. This optimized real-time assay will be useful as a new sensitive diagnostic tool in epidemiological investigations of diarrheas in cattle.