A rapid and sensitive PCR-based diagnostic assay to detect bovine herpesvirus 1 in routine diagnostic submissions

We describe a rapid, sensitive and specific polymerase chain reaction (PCR) assay for the detection of BHV1 DNA in a range of routine diagnostic submi ssions without the need for prior virus isolation. The assay, which is base d on the selected amplification of a portion of the viral tk gene, detected both BHV1.1 and BHV1.2 subtypes in a panel of 15 characterised field isola tes, and its sensitivity was estimated to be <0.125 TCID50. BHV2, alcephali ne herpesvirus, BHV4, equine herpesvirus 1 (EHV1), EHV4 and pseudorabies vi rus were not detected confirming the specificity of the assay. One hundred and five diagnostic submissions, including tissues, nasal secretions and na sal swabs were taken from cattle with respiratory disease and tested using the routine methods of virus isolation (VI) and the fluorescent antibody te st (FAT), and the results were compared with those obtained by PCR. The PCR assay detected BHV1 DNA in all samples that were positive by VI. BHV1 DNA was also detectable by PCR in raw and extended semen samples at a sensitivi ty of 1 TCID50 per 50 mu l. The assay also detected BHV5, permitting differ entiation between it and BHV1 by virtue of the size of the amplified PCR pr oduct. The PCR assay is more sensitive and independent of sample quality th an either virus isolation or FAT, and it is faster than virus isolation. Th e sample preparation method is simple with few steps involved. There are no extra post-amplification blotting/hybridisation steps and the assay is not based on a nested PCR strategy that might otherwise exacerbate the problem of oversensitivity/contamination in the routine use of such a test in a di agnostic laboratory. This assay would permit discrimination between those a nimals naturally infected with wild type BHV1 and those vaccinated with tk- BHV1 strains. (C) 2000 Elsevier Science B.V. All rights reserved.