Development and validation of a triplex real-time PCR assay for the rapid detection and differentiation of wild-type and glycoprotein E-deleted vaccine strains of Bovine herpesvirus type 1.

Two important components of control programs for Bovine herpesvirus type 1 (BoHV-1), a major pathogen of cattle, are the detection of outbreaks and vaccination with glycoprotein E (gE)-deleted marker vaccines. In addition to serology, rapid and accurate investigation of BoHV-1 and genetic differentiation of vaccine and wild-type strains are also important methods. Therefore, a triplex quantitative real-time polymerase chain reaction (qPCR) for testing BoHV-1 was developed. Apart from a BoHV-1 specific glycoprotein D (gD) assay, a gE-specific system for differentiation between wild-type BoHV-1 and gE-deleted vaccine strains was established. Finally, an internal control, based on the beta-actin gene was introduced successfully completing the multiplex system. The triplex BoHV-1-qPCR has an analytical sensitivity of less than 10 genome copies per reaction, and the diagnostic sensitivity was equal to or even greater than that of the 'gold standard' method of virus isolation in cell culture. A series of reference strains, including gE-deleted BoHV-1 and field isolates were detected reliably. For validation of the specificity of the test, nasal swabs, semen and different organ material from cattle, negative for BoHV-1, and genetically related herpesvirus strains were examined. The new multiplex BoHV-1-specific qPCR system allows highly sensitive and rapid genetic detection and differentiation of BoHV-1 and will be a valuable method for the control of BoHV-1 infection.