RESUMO
A duplex realtime PCR was developed and validated for the simultaneous detection of Brucella and bovine alphaherpesvirus1 (BoHV1) from bovine clinical specimens. The bcsp31 gene of Brucella and gB gene of BoHV1 were used as targets in the assay. The limit of detection for BoHV1 was 0.03 TCID50 of virus and 10 plasmid copies containing the target gene while for Brucella it was 4.1 × 101 CFUs. Intraassay and interassay values showed high repeatability and reproducibility of the assay. The diagnostic sensitivity (dsn) and diagnostic specificity (dsp) of the duplex assay were determined by screening 443 clinical specimens and comparing the results with the respective individual assays. The dsn and dsp for detection of Brucella were found to be 95.24% and 95.65%, respectively whereas for BoHV1, the dsn (100%) and dsp (99.47%) were slightly higher. The duplex assay had a very good degree of agreement with the respective individual realtime PCR test {kappa value 0.97 for Brucella and 0.95 for BoHV1}. The results of the current study suggest that the duplex assay would be a costeffective and timesaving alternative for the individual realtime PCR assay for the detection of Brucella and BoHV1.