A Sensitive and Specific PCR/dot Blot Hybridization assay for the Detection of Ovine Herpesvirus 2, a Gamma Herpesvirus

ABSTRACT

Polymerase chain reaction (PCR) provides a powerful technique for identifying viruses and studying the homology between viral nucleic acids. However, PCR assay has limitations in its susceptibility to contamination or to enzymatic inhibitors. In order to avoid problems related to nucleic acid amplification, efforts have been made to obtain specific hybridization assays, such as dot blot hybridization (DBH). DBH has higher specificity and lower sensitivity than PCR. The aims of the present study were to develop a sensitive and specific assay for the detection of ovine herpesvirus 2 (OvHV-2), a gamma herpesvirus. PCR/DBH assay for detecting OvHV-2 DNA was developed and evaluated for its sensitivity and specificity. OvHV-2 specific primer pairs, 755/556, were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 102 viral copies. For DBH, the amplified DNA with OvHV-2 specific primer pairs, 556/555, was labeled by the incorporation of digoxigenin (DIG). This DIGlabeled probe was capable of detecting 104 viral copies of purified OvHV-2 DNA by DBH. On the other hand, PCR/ DBH was more sensitive than either PCR or DBH and also very specific. The results showed that the sensitivity of PCR/DBH was higher and stronger than that of PCR and DBH alone. This PCR/DBH assay can be applied efficiently to confirm the presence of OvHV-2 virus on clinical samples and to differentiate specifically between OvHV-2 infection and other viral infections.