Nested PCR for ILTV DNA Detection

A nested PCR was used to test for the presence of ILTV DNA in TG collected on 21 dpv, TG co-culture supernatants and selected swabs collected on 21 dpv. Conjunctival mucosa, palatine cleft, infraorbital sinus and tracheal swabs were only tested for the presence of ILTV DNA using the nested PCR if the swabs collected 21 dpv tested negative for ILTV DNA using the less sensitive UL15 qPCR, and if the TG sample from the same individual bird was positive for ILTV using the nested PCR. This nested PCR, the universal herpes virus (UHV) nested PCR, targets a conserved region of the herpes virus DNA polymerase gene [38 (link)]. Products amplified during the second round of this PCR were purified using QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. Depending on the yield of purified DNA, amplicons were either directly subjected to sequencing or cloned into pGEM-T (Promega) following manufacturer’s instructions before sequencing. Sequencing reactions were performed using Big Dye Terminator v3.1 (Life Technologies Corporation, Carlsbad, USA) according to manufacturer’s instructions. Sample electrophoresis and sequencing was performed at the Centre for Translational Pathology, University of Melbourne. Geneious software version 9.1.3 [39 (link)] was used to analyse the sequencing data.