Development of species-specific PCR techniques for the detection of tortoise herpesvirus

Previously, a nested polymerase chain reaction (PCR) was employed with cons ensus degenerate primers targeting highly conserved motifs within herpesvir al DNA polymerase genes to detect a newly described tortoise herpesvirus. H owever, nucleotide, sequence information obtained from the final amplified fragment was restricted to a small region of 181 bp. In the present study, additional sequences flanking this segment were determined from a PCR produ ct successfully amplified using a set of known degenerate primers, which co vered a 692-bp region within the tortoise herpesviral DNA polymerase gene. Polymerase chain reaction primers for specific amplification of the tortois e herpesviral DNA were designed on the basis of these nucleotide sequences and successfully amplified tortoise herpesviral DNA from the tissues of tor toises that were well characterized histopathologically with herpesviral in fection. The lower limit of detection was 1,000 herpesviral DNA equivalents in the presence of normal tortoise genomic DNA. Furthermore, a more sensit ive and specific PCR technique for the identification of herpesviral infect ions in tortoises was developed employing a heminested form, which will ena ble the detection of latent infections or herpesvirus carriers in tortoises .