Evaluation of a specialized filter-paper matrix for transportation of extended bovine semen to screen for bovine herpesvirus-1 by real-time PCR

The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN2) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN2 related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA((R))) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method. A protocol for extraction of BoHV-1 DNA from FTA((R)) card spotted with extended semen was optimized. The viral DNA was found to be stable on FTA((R)). card for at least 28 days when the cards are stored at 4 degrees-37 degrees C. The analytical sensitivity for the assay was determined using variable dilutions of BoHV-1 spiked semen and positive plasmid harbouring gB gene (97bp) spotted onto FTA((R)) card and it was found to be 10(0.8) TCID50/ml or 100 copies respectively in real-time PCR. The test could detect as low as 10(0.008) TCID50/ml or 1 copy of positive plasmid when more number of replicates (n = 6) of the same sample were tested. This sensitivity was found to be comparable to Chelex method and both the methods demonstrated a very strong correlation (r = 0.9774; 95% CI: 0.9620-0.9860) in terms of Ct value (p < 0.0001). The diagnostic sensitivity and specificity of the FTA method in comparison to the Chelex method was 83.08% (95% CI: 71.73%-91.24%) and 93.23% (95% CI: 89.38%-96.01%) respectively when 316 samples were screened by both the methods. The degree of agreement between these two tests was good (Kappa value: 0.738; 95% CI: 0.646-0.829). The method was found to be robust and highly repeatable in inter-assay and intra-assay precision testing. The result suggests that the FTA((R)) card holds promise as an alternative system for transportation of EFS for downstream screening of BoHV-1 DNA.