Development of a multiplex polymerase chain reaction for the differentiation of bovine herpesvirus-1 and -5.

Bovine herpesvirus-1 (BHV-1) and bovine herpesvirus-5 (BHV-5) are closely related viruses which exhibit some important differences at the genetic and immunogenic levels which may explain the differences in their pathogenicity and epidemiological characteristics. A multiplex polymerase chain reaction (M-PCR) was developed to detect and differentiate between BHV-1 and BHV-5. In this M-PCR two pairs of primers (TK1, TK2 and GD1, GD2) were used in the same reaction mix to amplify a thymidine kinase genomic region (183 bp) of BHV-1 and one genomic region of the gLycoprotein D (564 bp) of BHV-5. The specificity of the M-PCR was demonstrated when using both primers pairs simultaneously with BHV-1 and BHV-5 templates. The two expected bands were amplified without the apparition of non-specific products. However, when other herpesvirus strains were used, there was no amplification. To evaluate the sensitivity of the assay, dilutions of purified viral DNA were made for M-PCR amplification. The detection limit was 7 pg for BHV-1 and 22 pg for BHV-5. It was also determined by comparing the M-PCR with viral isolation. M-PCR was able to detect one log10 more than viral isolation for BHV-1 and for BHV-5 was two logarithms lower. The applicability of M-PCR was demonstrated on different specimens. Twenty isolates from field samples (11 BHV-1 and nine BHV-5) were positive by M-PCR, and the results were completely coincident with previous characterization using the immunoperoxidase assay. M-PCR could detect viral DNA in organ samples from natural infections, such as semen and brain. In addition, M-PCR detected more positive samples than observation of the citophatic effect in cell culture of nasal swabs from experimentally infected animals in two different assays. Owing to the difference in size of the M-PCR products which allows easy identification in an electrophoretic run, it is not necessary to use extra blotting and hybridization steps or a second round of amplification to differentiate clearly between BHV-1 and BHV-5.