Detection of bovine pestiviruses in sera of beef calves by aRT-PCR based on a newly designed set of pan–bovine pestivirusprimers

摘要

The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2,respectively) and HoBi-like pestivirus (HoBiPeV) are importantpathogens of cattle, and a number of reverse-transcription PCR(RT-PCR)–based assays have been developed for their detection inclinical specimens. We evaluated a newly designed set of pan–bovinepestivirus primers (BP189-389) in a gel-based RT-PCR screening testfor pestiviruses in the sera of beef calves destined for export fromsouthern Brazil. Serum samples positive for BVDV antigens by anantigen ELISA ( = 135) were submitted to RT-PCRassays using different sets of primers, followed by nucleotidesequencing of the amplicons. RT-PCR with pestivirus primers 324-326detected 110 positive samples: BVDV-1 ( = 62),BVDV-2 ( = 38), and HoBiPeV ( =10). A PCR using primers HCV90-368 detected 97 positive samples (64BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2–specificprimers (2F-2R) detected 45 positive samples (including 38 detected byprimers 324-326 and 33 by HCV90-368); whereas a RT-PCR usingHoBiPeV-specific primers (N2-R5) detected 26 positive samples(including 10 detected by primers 324-326).The assay using the primersBP189-389 detected all 135 ELISA-positive samples, including the 26HoBiPeV detected by primers N2-R5. Our results demonstrated thatprimers BP189-389 compare favorably against other primer sets in thedetection of bovine pestiviruses, especially HoBiPeV. Thisconventional PCR may be useful for efficient detection of pestivirusesin bovine sera and other specimens as well, especially in laboratorieswithout real-time PCR equipment.